25934, a novel fatty acid desaturase and uses therefor

ABSTRACT

The invention provides isolated nucleic acids molecules, designated 25934 nucleic acid molecules, which encode a novel desaturase family member. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing 25934 nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a 25934 gene has been introduced or disrupted. The invention still further provides isolated 25934 proteins, fusion proteins, antigenic peptides and anti-25934 antibodies. Diagnostic methods utilizing compositions of the invention are also provided. The invention also provides methods for modulating fatty acid metabolism utilizing the compositions of the invention. Accordingly, methods of treating, preventing and/or diagnosing cardiovascular disorders, such atherosclerosis, hypertriglyceridemia, hypercholesterolemia, and hyperlipidemia, are disclosed.

RELATED APPLICATIONS

This application claims priority to U.S. provisional application No.60/187,455 filed on Mar. 7, 2000, the contents of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Fatty acid desaturases are critical regulatory enzymes of unsaturatedfatty acid biosynthesis and catalyze the conversion of a single bondbetween two carbon atoms (C—C) to a double bond (C═C) in a fatty acylchain. The resultant double bond is often referred to as an unsaturatedbond. Eukaryotic fatty acid desaturases, typically, are iron-containingenzymes that catalyze the NAD-(P)H and O₂-dependent introduction ofdouble bonds into methylene-interrupted fatty acid chains. Examinationof the deduced amino acid sequence from mammals, fungi, insects, higherplants and cyanobacteria has revealed three regions of conserved primarysequence containing HX(3 or 4)H, HX(2 or 3), and HX(2 or 3)HH. Thismotif is also present in the bacterial membrane enzymes alkalinehydroxylase (omega-hydroxylase) and xylene monooxygenase.

There are three types of eukaryotic fatty acid desaturases, acyl-CoA,acyl-ACP, and acyl-lipid desaturases (Ntambi et al., Biochem. andBiophys. Res. Com. 266:1-4, 1999). In plants and cyanobacteria,acyl-lipid desaturases catalyzing most desaturation reactions andintroduce unsaturated bonds into fatty acids that are in a lipid-boundform. Acyl-ACP desaturases are present in the plastids of plant cellsand insert a double bond into fatty acids that are bound to acyl carrierprotein (ACP). In animals, yeast and fungal cells, Acyl-CoA introduceunsaturated bonds into fatty acids that are bound to coenzyme A (CoA). Agene cloned from this family is stearoyl-CoA desaturase and this genehas been identified in many organisms including mice, rats, humans,yeast, ovines, and hamsters.

Fatty acid desaturases can introduce an unsaturated bond at a specificposition in a fatty acyl chain, for example, at the Δ6, Δ9, or Δ12position. Desaturases are typically integral membrane proteins inducedin the endoplasmic reticulum by dietary manipulations and then rapidlydegraded (Ozols, J. (1997) MBC Vol. 8 (11): 2281-2290). Unsaturatedfatty acids can be formed from a variety of fatty acids includingpalmitate and stearate resulting in the formation of unsaturated fattyacids palmitoleate (16:1), and oleate (18:1).

In mammals, the rate limiting step in the biosynthesis ofmonounsaturated fatty acids is the insertion of an unsaturated bond bystearoyl-CoA desaturase (SCD) in the Δ9 position of the fatty acid. SCDpreferentially catalyzes the synthesis of oleic acid. Oleate enrichedlow density lipoprotein (LDL) exhibits increased affinity for the vesselwall, and is therefore pro-atherogenic (Rudel, L. L. et al. (1997) J.Clin. Invest. 1:100(1):74-83). SCD involvement in generating atherogenicLDL variants and in regulating triglyceride synthesis is furthersupported by the finding that polyunsaturated fatty acids (PUFA), whichprotect against atherosclerosis, negatively regulate the expression ofthe SCD gene (Rudel, L L et al. (1995) Atheroscler. Thromb. Vasc. Biol.15(12):2101-10; Ntambi, J M (1999) J. Lipid Res. 40(9):1549-58).Moreover, a mouse deficient for SCD exhibits significant reduction intriglycerides (Miyazaki, M. et al. (2000) J. Biol. Chem, in press).

Unsaturated fatty acids play an important role in normal and diseasedorganisms. For example, the degree of fatty acid unsaturation in cellmembrane lipids determines membrane fluidity. Moreover, the productionof monounsaturated fatty acids, which once complexed with lipoproteinssuch as LDL, show increased affinity for the vessels wall has profoundimplications for cardiovascular disorders caused by aberrant fatty acidmetabolism. Examples of such disorders include atherosclerosis,hypertriglyceridemia, hypercholesterolemia, hyperlipidemia, amongothers.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery of a novelfatty acid desaturase, referred to herein as “25934”. The nucleotidesequence of a cDNA encoding 25934 is shown in SEQ ID NO:1, and the aminoacid sequence of a 25934 polypeptide is shown in SEQ ID NO:2. Inaddition, the nucleotide sequences of the coding region are depicted inSEQ ID NO:3.

Accordingly, in one aspect, the invention features a nucleic acidmolecule which encodes a 25934 protein or polypeptide, e.g., abiologically active portion of the 25934 protein. In a preferredembodiment the isolated nucleic acid molecule encodes a polypeptidehaving the amino acid sequence of SEQ ID NO:2. In other embodiments, theinvention provides isolated 25934 nucleic acid molecules having thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or the sequenceof the DNA insert of the plasmid deposited with ATCC Accession Number2167. In still other embodiments, the invention provides nucleic acidmolecules that are substantially identical (e.g., naturally occurringallelic variants) to the nucleotide sequence shown in SEQ ID NO:1, SEQID NO:3, or the sequence of the DNA insert of the plasmid deposited withATCC Accession Number 2167. In other embodiments, the invention providesa nucleic acid molecule which hybridizes under stringent hybridizationconditions to a nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO:1 or 3, or the sequence of the DNA insert of the plasmiddeposited with ATCC Accession Number 2167, wherein the nucleic acidencodes a full length 25934 protein or an active fragment thereof.

In a related aspect, the invention further provides nucleic acidconstructs which include a 25934 nucleic acid molecule described herein.In certain embodiments, the nucleic acid molecules of the invention areoperatively linked to native or heterologous regulatory sequences. Alsoincluded, are vectors and host cells containing the 25934 nucleic acidmolecules of the invention e.g., vectors and host cells suitable forproducing 25934 nucleic acid molecules and polypeptides.

In another related aspect, the invention provides nucleic acid fragmentssuitable as primers or hybridization probes for the detection of25934-encoding nucleic acids.

In still another related aspect, isolated nucleic acid molecules thatare antisense to a 25934 encoding nucleic acid molecule are provided.

In another aspect, the invention features, 25934 polypeptides, andbiologically active or antigenic fragments thereof that are useful,e.g., as reagents or targets in assays applicable to treatment anddiagnosis of 25934 mediated or related disorders, e.g., a cardiovasculardisorder. In another embodiment, the invention provides 25934polypeptides having a 25934 activity. Preferred polypeptides are 25934proteins including at least one desaturase domain, and, preferably,having a 25934 activity, e.g., a 25934 activity as described herein.

In other embodiments, the invention provides 25934 polypeptides, e.g., a25934 polypeptide having the amino acid sequence shown in SEQ ID NO:2;the amino acid sequence encoded by the cDNA insert of the plasmiddeposited with ATCC Accession Number 2167; an amino acid sequence thatis substantially identical to the amino acid sequence shown in SEQ IDNO:2; or an amino acid sequence encoded by a nucleic acid moleculehaving a nucleotide sequence which hybridizes under stringenthybridization conditions to a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1 or 3, or the sequence of the DNAinsert of the plasmid deposited with ATCC Accession Number 2167, whereinthe nucleic acid encodes a full length 25934 protein or an activefragment thereof.

In a related aspect, the invention further provides nucleic acidconstructs which include a 25934 nucleic acid molecule described herein.

In a related aspect, the invention provides 25934 polypeptides orfragments operatively linked to non-25934 polypeptides to form fusionproteins.

In another aspect, the invention features antibodies and antigen-bindingfragments thereof, that react with, or more preferably specifically bind25934 polypeptides.

In another aspect, the invention provides methods of screening forcompounds that modulate the expression or activity of the 25934polypeptides or nucleic acids.

In still another aspect, the invention provides a process for modulating25934 polypeptide or nucleic acid expression or activity, e.g. using thescreened compounds. In certain embodiments, the methods involvetreatment or prophylaxis of conditions related to activity or expressionof the 25934 polypeptides or nucleic acids, such as cardiovascular,neurological, metabolic, reproductive (e.g., ovarian), renal and hepaticdisorders.

Examples of cardiovascular disorders include e.g., atherosclerosis,thrombosis, heart failure, ischemic heart disease, angina pectoris,myocardial infarction, sudden cardiac death, hypertensive heart disease;non-coronary vessel disease, such as arteriolosclerosis, small vesseldisease, nephropathy, hypertriglyceridemia, hypercholesterolemia,hyperlipidemia, xanthomatosis, asthma, hypertension, emphysema andchronic pulmonary disease; or a cardiovascular condition associated withinterventional procedures (“procedural vascular trauma”), such asrestenosis following angioplasty, placement of a shunt, stet, stent,synthetic or natural excision grafts, indwelling catheter, valve orother implantable devices.

In a preferred embodiment, the cardiovascular disorder is caused byaberrant fatty acid metabolism. Examples of disorders involving aberrantfatty acid metabolism include, but are not limited to, atherosclerosis,arteriolosclerosis, hypertriglyceridemia, obesity, diabetes,hypercholesterolemia, xanthomatosis, and hyperlipidemia. Mostpreferable, the disorder is atherosclerosis.

In the cardiovascular applications, the agent is administered alone orin combination with a cholesterol lowering agent. Examples ofcholesterol lowering agents include bile acid sequestering resins (e.g.colestipol hydrochloride or cholestyramine), fibric acid derivatives(e.g. clofibrate, fenofibrate, or gemfibrozil), thiazolidenediones (e.g.troglitazone), or hydroxymethylglutaryl coenzyme A reductase (HMG-CoAreductase) inhibitors (e.g. statins, such as fluvastatin sodium,lovastatin, pravastatin sodium, or simvastatin), an ApoAII-loweringagent, a VLDL lowering agent, an ApoAI-stimulating agent, as well asinhibitors of, nicotinic acid, niacin, or probucol. Preferredcholesterol lowering agents include inhibitors of HMG-CoA reductase(e.g., statins), nicotinic acid, and niacin.

The cholesterol lowering agent can be administered prior to, at the sametime, or after administration of the agent, in single or multipleadministration schedules. For example, the cholesterol lowering agentand the agents of the invention can be administered continually over apreselected period of time, or administered in a series of spaced doses,i.e., intermittently, for a period of time.

In a preferred embodiments, the agent, alone or in combination with thecholesterol lowering agent, inhibit (block or reduce) atheroscleroticlesion formation or development, e.g., so as to inhibit lipidaccumulation, increase plaque stability or promote lesion regression.

In still another aspect, the invention features a method of modulating(e.g., enhancing or inhibiting) fatty acid metabolism, in a subject. Themethod includes administering to the subject an agent that modulates theactivity or expression of a 25934 polypeptide or nucleic acid, in anamount effective to modulate the conversion of saturated fatty acids tounsaturated fatty acids, e.g., monounsaturated fatty acids.

In a preferred embodiment, the 25934 polypeptide has an amino acidsequence identical to, or substantially identical to, SEQ ID NO:2. Inother embodiments, the 25934 polypeptide is a fragment of at least 15,20, 50, 100, 150, 200, 250, 300 or more contiguous amino acids of SEQ IDNO:2.

In a preferred embodiment, the 25934 nucleic acid has a nucleotidesequence identical to, or substantially identical to, SEQ ID NO:1 or 3.In other embodiments, the 25934 nucleic acid is a fragment of at least50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 ormore contiguous nucleotides of SEQ ID NO:1 or 3.

In a preferred embodiment, the agent modulates, e.g., decreases orblocks, desaturase activity or expression. For example, the agent (e.g.,a polyunsaturated fatty acid, or nicotinic acid or niacin) maynegatively regulate 25934 protein expression.

In a preferred embodiment, the agent modulates (e.g., decreases orincreases) the activity or expression of a 25934 polypeptide or nucleicacid such that modulation of one or more of: lipoprotein composition,lipid (e.g., triglyceride) levels, apolipoprotein B, total cholesterol,or lipoprotein (a), occurs. For example, the agent can decrease lowdensity lipoprotein (LDL)-cholesterol levels, and/or lipid (e.g.,triglyceride) levels. Preferably, the agent can also increasehigh-density lipoprotein (HDL)-cholesterol levels.

In a preferred embodiment, the agent modulates (e.g., increases ordecreases) expression of the 25934 nucleic acid by, e.g., modulatingtranscription, mRNA stability, etc.

In preferred embodiments, the agent is a peptide, a phosphopeptide, asmall molecule, e.g., a member of a combinatorial or natural productlibrary, or an antibody, or any combination thereof.

In additional preferred embodiments, the agent is an antisense, aribozyme, or a triple helix molecule, or a 25934 nucleic acid or afragment thereof, or any combination thereof.

In a preferred embodiment, the subject is a patient undergoing atherapeutic or prophylactic protocol. Preferably, the subject is a humansuffering from, or at risk of a cardiovascular disease, e.g.,atherosclerosis, thrombosis, heart failure, ischemic heart disease,angina pectoris, myocardial infarction, sudden cardiac death,hypertensive heart disease; non-coronary vessel disease, such asarteriolosclerosis, small vessel disease, nephropathy,hypertriglyceridemia, obesity, diabetes, hypercholesterolemia,hyperlipidemia, xanthomatosis, asthma, hypertension, emphysema andchronic pulmonary disease; or a cardiovascular condition associated withinterventional procedures (“procedural vascular trauma”), such asrestenosis following angioplasty, placement of a shunt, stet, stent,synthetic or natural excision grafts, indwelling catheter, valve orother implantable devices.

In a preferred embodiment, the subject is a human suffering from, or atrisk of a disorder involving aberrant fatty acid metabolism. Examples ofsuch disorders include, but are not limited to, atherosclerosis,arteriolosclerosis, hypertriglyceridemia, obesity, diabetes,hypercholesterolemia, xanthomatosis and hyperlipidemia. Most preferable,the disorder is atherosclerosis.

In other embodiments, the subject is a non-human animal, e.g., anexperimental animal.

In a preferred embodiment, the agent is administered alone or incombination with a cholesterol lowering agent. Examples of cholesterollowering agents include bile acid sequestering resins (e.g. colestipolhydrochloride or cholestyramine), fibric acid derivatives (e.g.clofibrate, fenofibrate, or gemfibrozil), thiazolidenediones (e.g.troglitazone), or hydroxymethylglutaryl coenzyme A reductase (HMG-CoAreductase) inhibitors (e.g. statins, such as fluvastatin sodium,lovastatin, pravastatin sodium, or simvastatin), an ApoAII-loweringagent, a VLDL lowering agent, an ApoAI-stimulating agent, as well asinhibitors of, nicotinic acid, niacin, or probucol. Preferredcholesterol lowering agents include inhibitors of HMG-CoA reductase(e.g., statins), nicotinic acid, and niacin.

The cholesterol lowering agent can be administered prior to, at the sametime, or after administration of the agent, in single or multipleadministration schedules. For example, the cholesterol lowering agentand the agents of the invention can be administered continually over apreselected period of time, or administered in a series of spaced doses,i.e., intermittently, for a period of time.

In a preferred embodiment, the agent, alone or in combination with thecholesterol lowering agent, inhibits (blocks or reduces) atheroscleroticlesion formation or development, e.g., so as to inhibit lipidaccumulation, increase plaque stability or promote lesion regression.

In a preferred embodiment, the agent, administered alone or incombination with the cholesterol lowering agent, results in a favorableplasma lipid profile (e.g., increased HDL and/or reduced LDL).

In yet another aspect, the invention features a method of treating orpreventing a cardiovascular disorder (e.g., atherosclerosis), in asubject. The method includes administering to the subject an agent thatmodulates the activity or expression of a 25934 polypeptide or nucleicacid, in an amount effective to treat or prevent the cardiovasculardisorder.

In a preferred embodiment, the 25934 polypeptide has an amino acidsequence identical to, or substantially identical to, SEQ ID NO:2. Inother embodiments, the 25934 polypeptide is a fragment of at least 15,20, 50, 100, 150, 200, 250, 300 or more contiguous amino acids of SEQ IDNO:2.

In a preferred embodiment, the 25934 nucleic acid has a nucleotidesequence identical to, or substantially identical to, SEQ ID NO:1 or 3.In other embodiments, the 25934 nucleic acid is a fragment of at least50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 ormore contiguous nucleotides of SEQ ID NO:1 or 3.

In a preferred embodiment, the agent modulates, e.g., decreases orblocks, desaturase activity or expression. For example, the agent (e.g.,a polyunsaturated fatty acid, or nicotinic acid or niacin) maynegatively regulate 25934 protein expression.

In a preferred embodiment, the agent modulates (e.g., decreases orincreases) the activity or expression of a 25934 polypeptide or nucleicacid such that modulation of one or more of: lipoprotein composition,lipid (e.g., triglyceride) levels, apolipoprotein B, total cholesterol,or lipoprotein (a), occurs. For example, the agent can decrease lowdensity lipoprotein (LDL)-cholesterol levels, and/or lipid (e.g.,triglyceride) levels. Preferably, the agent can also increasehigh-density lipoprotein (HDL)-cholesterol levels.

In a preferred embodiment, the agent modulates (e.g., increases ordecreases) expression of the 25934 nucleic acid by, e.g., modulatingtranscription, mRNA stability, etc.

In preferred embodiments, the agent is a peptide, a phosphopeptide, asmall molecule, e.g., a member of a combinatorial or natural productlibrary, or an antibody, or any combination thereof.

In additional preferred embodiments, the agent is an antisense, aribozyme, or a triple helix molecule, or a 25934 nucleic acid or afragment thereof, or any combination thereof.

In a preferred embodiment, the subject is a patient undergoing atherapeutic or prophylactic protocol. Preferably, the subject is a humansuffering from, or at risk of a cardiovascular disease, e.g.,atherosclerosis, thrombosis, heart failure, ischemic heart disease,angina pectoris, myocardial infarction, sudden cardiac death,hypertensive heart disease; non-coronary vessel disease, such asarteriolosclerosis, small vessel disease, nephropathy,hypertriglyceridemia, hypercholesterolemia, hyperlipidemia,xanthomatosis, asthma, hypertension, emphysema and chronic pulmonarydisease; or a cardiovascular condition associated with interventionalprocedures (“procedural vascular trauma”), such as restenosis followingangioplasty, placement of a shunt, stet, stent, synthetic or naturalexcision grafts, indwelling catheter, valve or other implantabledevices.

In a preferred embodiment, the subject is a human suffering from, or atrisk of a disorder involving aberrant fatty acid metabolism. Examples ofsuch disorders include, but are not limited to, atherosclerosis,arteriolosclerosis, hypertriglyceridemia, obesity, diabetes,hypercholesterolemia, xanthomatosis and hyperlipidemia. Most preferable,the disorder is atherosclerosis.

In other embodiments, the subject is a non-human animal, e.g., anexperimental animal.

In a preferred embodiment, the agent is administered alone or incombination with a cholesterol lowering agent. Examples of cholesterollowering agents include bile acid sequestering resins (e.g. colestipolhydrochloride or cholestyramine), fibric acid derivatives (e.g.clofibrate, fenofibrate, or gemfibrozil), thiazolidenediones (e.g.troglitazone), or hydroxymethylglutaryl coenzyme A reductase (HMG-CoAreductase) inhibitors (e.g. statins, such as fluvastatin sodium,lovastatin, pravastatin sodium, or simvastatin), an ApoAII-loweringagent, a VLDL lowering agent, an ApoAI-stimulating agent, as well asinhibitors of, nicotinic acid, niacin, or probucol. Preferredcholesterol lowering agents include inhibitors of HMG-CoA reductase(e.g., statins), nicotinic acid, and niacin.

The cholesterol lowering agent can be administered prior to, at the sametime, or after administration of the agent in single or multipleadministration schedules. For example, the cholesterol lowering agentand the agents of the invention can be administered continually over apreselected period of time, or administered in a series of spaced doses,i.e., intermittently, for a period of time as a therapeutic orpreventative measure.

In a preferred embodiment, the agent, alone or in combination with thecholesterol lowering agent, inhibits (blocks or reduces) atheroscleroticlesion formation or development, e.g., so as to inhibit lipidaccumulation, increase plaque stability or promote lesion regression.

In a preferred embodiment, the agent, administered alone or incombination with the cholesterol lowering agent, results in a favorableplasma lipid profile (e.g., increased HDL and/or reduced LDL).

The invention also features a method of diagnosing a disorder, e.g., acardiovascular disorder (e.g., atherosclerosis), in a subject. Themethod includes evaluating the expression or activity of a 25934 nucleicacid or a 25934 polypeptide, such that, a difference in the level of25934 nucleic acid or 25934 polypeptide relative to a normal subject ora cohort of normal subjects is indicative of the disorder. Because 25934is regulated by Niacin (a clinically used therapeutic), 25934 levelsand/or activity may be considered a marker for Niacin activity and orefficacy.

In a preferred embodiment, the subject is a human.

In a preferred embodiment, the evaluating step occurs in vitro or exvivo. For example, a sample, e.g., a blood sample, is obtained from thesubject.

In a preferred embodiment, the evaluating step occurs in vivo. Forexample, by administering to the subject a detectably labeled agent thatinteracts with the 25934 nucleic acid or polypeptide, such that a signalis generated relative to the level of activity or expression of the25934 nucleic acid or polypeptide.

In a preferred embodiment, the disorder is a cardiovascular disorder,e.g., a cardiovascular disorder as described herein.

In a preferred embodiment, the disorder is atherosclerosis.

The invention also provides assays for determining the activity of orthe presence or absence of 25934 polypeptides or nucleic acid moleculesin a biological sample, including for disease diagnosis.

In a further aspect, the invention provides assays for determining thepresence or absence of a genetic alteration in a 25934 polypeptide ornucleic acid molecule, including for disease diagnosis, predictingtriglyceride levels, predicting response to Niacin, and predicting aresponse to triglyceride therapy.

In yet another aspect, the invention features a method for identifyingan agent, e.g., a compound, which modulates the activity of a 25934polypeptide, e.g., a 25934 polypeptide as described herein, or theexpression of a 25934 nucleic acid, e.g., a 25934 nucleic acid asdescribed herein, including contacting the 25934 polypeptide or nucleicacid with a test agent (e.g., a test compound); and determining theeffect of the test compound on the activity of the polypeptide ornucleic acid to thereby identify a compound which modulates the activityof the polypeptide or nucleic acid. Such agents are useful for treatingor preventing a 25934-mediated disorders, e.g., cardiovascular disorders(e.g., atherosclerosis) or metabolic disorders (e.g., obesity ordiabetes).

In a preferred embodiment, the contacting step occurs in vitro or exvivo. For example, a sample, e.g., a blood sample, is obtained from thesubject.

In a preferred embodiment, the contacting step occurs in vivo. Forexample, by administering to the subject a detectably labeled agent thatinteracts with the 25934 nucleic acid or polypeptide, such that a signalis generated relative to the level of activity or expression of the25934 nucleic acid or polypeptide.

In a preferred embodiment, the activity of the 25934 polypeptide isprotein desaturase activity, e.g., catalyzing the conversion ofsaturated fatty acids to unsaturated fatty acids. In such embodiment,the 25934 polypeptide is contacted with a saturated fatty acid, e.g.,oleic acid, palmitate and stearate.

In a preferred embodiment, the agent is an inhibitor (partial orcomplete inhibitor) of 25934 polypeptide activity or expression.

In a preferred embodiment, the agent modulates one or more of:lipoprotein composition, lipid (e.g., triglyceride) levels, LDL, HDL,apolipoprotein B, total cholesterol, or lipoprotein (a). For example,the agent can decrease low density lipoprotein (LDL)-cholesterol levels,and/or lipid (e.g., triglyceride) levels. Preferably, the agent can alsoincrease high-density lipoprotein (HDL)-cholesterol levels.

In preferred embodiments, the agent is a peptide, a phosphopeptide, asmall molecule, e.g., a member of a combinatorial library, or anantibody, or any combination thereof.

In additional preferred embodiments, the agent is an antisense, aribozyme, a triple helix molecule, or a 25934 nucleic acid, or anycombination thereof.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B depict a cDNA sequence (SEQ ID NO:1) and predicted amino acidsequence (SEQ ID NO:2) of human 25934. The methionine-initiated openreading frame of human 25934 (without the 5′ and 3′ untranslatedregions) which occurs at nucleotide 403 through 1392 of SEQ ID NO:1(shown also as coding sequence (SEQ ID NO:3).

FIG. 2 depicts a hydropathy plot of human 25934. Relative hydrophobicresidues are shown above the dashed horizontal line, and relativehydrophilic residues are below the dashed horizontal line. The cysteineresidues (cys) and an N-glycosylation site are indicated by shortvertical lines just below the hydropathy trace. The transmembraneregions are indicated by horizontal lines above the hydropathy trace.The numbers corresponding to the amino acid sequence of human 25934 areindicated. Polypeptides of the invention include 25934 fragments whichinclude: all or part of a hydrophobic sequence (a sequence above thedashed line, e.g., all or part of the sequence from about residue 71 toabout residue 91 of SEQ ID NO:2; all or part of a hydrophilic fragment(e.g., a fragment below the dashed line). Other fragments include acysteine or a glycosylation site.

FIG. 3 depicts an alignment of the desaturase domain of human 25934 witha consensus amino acid sequence derived from a hidden Markov model usingPFAM. The upper sequence is the consensus amino acid sequence (SEQ IDNO:4), while the lower amino acid sequence corresponds to amino acids 51to 295 of SEQ ID NO:2.

FIG. 4 is a bar graph depicting relative 25934 mRNA expression asdetermined by TaqMan assays on mRNA derived from the human fetal heart,spinal cord, brain (cortex, hypothalamus, glial cells), ovary, kidney,liver, endothelial cells and smooth muscle cells (SMC). Numbers on the Xaxis of the bar graph correspond to the following tissues: 1) AortaNormal; 2) Fetal Heart; 3) Heart Normal; 4) Heart CHF; 5) Vein Normal;6) Spinal Cord; 7) Brain Cortex; 8) Brain Hypothalamus; 9) Glial CellAstroc; 10) Brain Glioblast; 11) Breast Normal; 12) Breast Tumor; 13)Ovary Normal; 14) Ovary Tumor; 15) Pancreas; 16) Prostate; 17) Prostate;18) Colon Normal; 19) Colon Tumor; 20) Colon IBD; 21) Kidney Normal; 22)Liver Normal; 23) Liver Fibrosis; 24) Fetal Liver No; 25) Lung Normal;26) Lung Tumor; 27) Lung COPD; 28) Spleen Normal; 29) Tonsil Normal; 30)Lymph Node; 31) Thymus Normal; 32) Epithelial Cells; 33) EndothelialCells; 34) Skeletal Muscle; 35) Fibroblast; 36) Skin Normal; 37) AdiposeNormal; 38) Osteoblast; 39) Osteoblast; 40) Osteoblast Diff; 41)Osteoclasts; 42) Aortic SMC; 43) Aortic SMC; 44)Shear HUVEC; 45) StaticHUVEC; and 46) BM MNC. The highest 25934 mRNA expression, i.e., greaterthan 200 relative units, was observed in spinal cord, brain and ovary.High level mRNA expression, i.e., greater than 100 relative units wasobserved in the kidney, endothelial cells and human umbilical veinendothelial cells (HUVEC). Expression in liver was positive albeit low.

FIG. 5 is a bar graph depicting relative 25934 mRNA expression asdetermined by TaqMan assays on mRNA derived from an array of human livertissues from individual donors.

FIG. 6 depicts a comparison of the expression of 25934 mRNA and stearoylCoA desaturase (SCD) mRNA in a human liver samples from individualdonors. Expression of 25934 mRNA in human liver is equivalent to therelative expression of the SCD gene.

FIG. 7 is a bar graph depicting the inhibition of 25934 mRNA (viaTaqMan) expression in the marmoset animal model. Niacin treatment in themarmoset model results in significant repression of 25934 in the liver.

FIG. 8 is a multiple sequence alignment of the 25934 amino acid sequencewith the human (SEQ ID NO:7), rat (SEQ ID NO:8), and chicken (SEQ IDNO:9) delta-9 desaturase proteins. A comparison of the full length aminoacid sequence revealed 63.6%, 58.4%, and 58.4% identity between the25934 amino acid sequence and the chicken, human and rat amino acidsequences, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The human 25934 sequence (FIGS. 1A-B; SEQ ID NO:1), which isapproximately 1512 nucleotides long including untranslated regions,contains a predicted methionine-initiated coding sequence of about 990nucleotides (nucleotides 403-1392 of SEQ ID NO:1; SEQ ID NO:3). Thecoding sequence encodes a 330 amino acid protein (SEQ ID NO:2).

Human 25934 contains the following regions or other structural features:a desaturase domain located at about amino acid residues 51 to 295 ofSEQ ID NO:2; two transmembrane regions at about amino acids 50-93 and194-235 of SEQ ID NO:2; three cytoplasmic domains at about amino acids1-49, 94-193, and 236-330 of SEQ ID NO:2; one predicted N-glycosylationsite (PS00001) at about amino acids 233 to 236 of SEQ ID NO:2; onepredicted cAmp and cGMP dependent protein kinase phosphorylation site(PS00004) at about amino acids 311 to 314 of SEQ ID NO:2; four predictedProtein Kinase C phosphorylation sites (PS00005) at about amino acids 98to 100, 101 to 103, 255 to 257 and 308 to 310 of SEQ ID NO:2; twopredicted Casein Kinase II phosphorylation sites (PS00006) located atabout amino acids 138 to 141 and 283-286 of SEQ ID NO:2; four predictedN-myristoylation sites (PS00008) from about amino acids 23 to 28, 40 to45, 59 to 64, and 88 to 93 of SEQ ID NO:2; one predicted amidation site(PS00009) from about amino acid 170 to 173 of SEQ ID NO:2; and onepredicted fatty acid desaturase family 1 signature (PS00476) from aboutamino acid 268 to 282 of SEQ ID NO:2.

For general information regarding PFAM identifiers, PS prefix and PFprefix domain identification numbers, refer to Sonnhammer et al. (1997)Protein 28:405-420 andhttp://www.psc.edu/general/software/packages/pfam/pfam.html.

A plasmid containing the nucleotide sequence encoding human 25934 wasdeposited with American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va. 20110-2209, on Jun. 27, 2000 and assignedAccession Number 2167. This deposit will be maintained under the termsof the Budapest Treaty on the International Recognition of the Depositof Microorganisms for the Purposes of Patent Procedure. This deposit wasmade merely as a convenience for those of skill in the art and is not anadmission that a deposit is required under 35 U.S.C. §112.

The 25934 protein contains a significant number of structuralcharacteristics in common with members of the desaturase family. Theterm “family” when referring to the protein and nucleic acid moleculesof the invention means two or more proteins or nucleic acid moleculeshaving a common structural domain or motif and having sufficient aminoacid or nucleotide sequence homology as defined herein. Such familymembers can be naturally or non-naturally occurring and can be fromeither the same or different species. For example, a family can containa first protein of human origin as well as other distinct proteins ofhuman origin, or alternatively, can contain homologues of non-humanorigin, e.g., rat or mouse proteins. Members of a family can also havecommon functional characteristics.

Based on sequence homology, 25934 polypeptide is predicted to be amember of the desaturase family of enzymes, specifically the stearoyl-Codesaturase family (SCD family, EC 1.14.99.5) (see FIGS. 3 and 8). Theseenzymes are structurally and functionally homologous to one another, andcan convert a single bond to a double bond in a fatty acyl chain. SCDenzymes utilize oxygen and electrons from cytochrome b₅ for catalysis.Similar to other enzymes such as ribonucleotide reductases and methanemonoxygenases, stearoyl-CoA desaturases can have a conserved ironbinding motif which includes eight histidines (Shanklin et al. (1997)Proc. Natl. Acad. Sci. USA 94:2981-2986),“H-X(3-4)-H-X(7-41)-H-X(2-3)-H-H-X(61-189)-H-X(2-3)-H-H (SEQ ID NO:5).”The eight histidine residues common to desaturase family members aretypically divided among three regions of the protein: region Ia(H-X(3-4)-H); region Ib (the first H-X(2-3)-H-H sequence); and region II(the second H-X(2-3)-H-H sequence) (Shanklin et al. (1994) Biochemistry33:12787-94).

SCDs typically contain two or three long hydrophobic domains termed“transmembrane regions,” each of which is capable of spanning themembrane two times (Shanklin et al. (1994) Biochemistry 33:12787-94).Because a transmembrane region is capable of traversing the membranetwice, amino acid residues flanking a transmembrane region reside on thesame side of the membrane (Stukey et al. (1990) J. Biol. Chem.265:20144-49). Thus, when region I (regions Ia and Ib) and region II aredivided by a transmembrane region in a desaturase family member, theregions will typically reside on the same side of the membrane, e.g.,the cytoplasmic face of the endoplasmic reticulum membrane.

A 25934 polypeptides include a “desaturase domain” or regions homologouswith a “desaturase domain”. As used herein, the term “desaturase domain”includes an amino acid sequence of about 25 to 600 amino acid residuesin length and having a bit score for the alignment of the sequence tothe fatty acid desaturase domain (HMM) of at least 50. Preferably, adesaturase domain includes at least about 50-500 amino acids, morepreferably about 100-400 amino acid residues, or about 200-250 aminoacids and has a bit score for the alignment of the sequence to thedesaturase domain (HMM) of at least 60, 80, 100, 150, 200, 250, 300,450, 500 or greater. An alignment of the desaturase domain (amino acids51 to 295 of SEQ ID NO:2) of human 25934 with a consensus amino acidsequence derived from a hidden Markov model is depicted in FIG. 3.

In a preferred embodiment, 25934 polypeptide or protein has a“desaturase domain” or a region which includes at least about 50-500amino acids, more preferably about 100-400 amino acid residues, or about200-250 amino acid residues and has at least about 70% 80% 90% 95%, 99%,or 100% homology with a “desaturase domain,” e.g., the desaturase domainof human 25934 (e.g., residues 51-295 of SEQ ID NO:2). Preferably, thedesaturase domain of a 25934 polypeptide includes at least one, two,three, four, five, six, seven and preferably eight conserved histidines.Preferably, the histidines form an eight-histidine motif, which bindstwo iron atoms in the catalytic center. For example, a 25934 polypeptidecontains histidine residues at about amino acids 94, 99, 131, 134, 135,272, 275, and 276 of SEQ ID NO:2.

To identify the presence of a “desaturase” domain in a 25934 proteinsequence, and make the determination that a polypeptide or protein ofinterest has a particular profile, the amino acid sequence of theprotein can be searched against a database of HMMs (e.g., the Pfamdatabase, release 2.1) using the default parameterssanger.ac.uk/Software/Pfam/HMM_search). For example, the hmmsf program,which is available as part of the HMMER package of search programs, is afamily specific default program for MILPAT0063 and a score of 15 is thedefault threshold score for determining a hit. Alternatively, thethreshold score for determining a hit can be lowered (e.g., to 8 bits).A description of the Pfam database can be found in Sonhammer et al.(1997) Proteins 28(3):405-420 and a detailed description of HMMs can befound, for example, in Gribskov et al.(1990) Meth. Enzymol. 183:146-159;Gribskov et al.(1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh etal.(1994) J. Mol. Biol. 235:1501-1531; and Stultz et al.(1993) ProteinSci. 2:305-314, the contents of which are incorporated herein byreference. A search was performed against the HMM database resulting inthe identification of a “desaturase domain” domain in the amino acidsequence of human 25934 at about residues 51-295 of SEQ ID NO:2 (seeFIGS. 1A-B).

A 25934 family member includes a desaturase domain and optionally also afatty acid desaturase family 1 signature, i.e., a motif that matches theProSite motif PS00476, “G-E-X-[FY]-H-N-[FY]-H-H-X-F-P-X-D-Y (SEQ IDNO:6),” e.g., the peptide sequence “GEGFHNYHTFPFDY” located at aboutresidues 268 to 282 of SEQ ID NO:2.

In one embodiment, a 25934 protein includes at least one, preferablytwo, transmembrane regions. As used herein, the term “transmembraneregion” includes an amino acid sequence of about 20 amino acid residuesin length that spans a phospholipid membrane, e.g., an endoplasmicreticulum membrane, twice. More preferably, a transmembrane regionincludes about at least 22, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, or70 amino acid residues and spans a phospholipid membrane twice.Transmembrane regions are rich in hydrophobic residues, and typicallyhave an a-helical structure. In a preferred embodiment, at least 50%,60%, 70%, 80%, 90%, 95% or more of the amino acids of a transmembranedomain are hydrophobic, e.g., leucines, isoleucines, tyrosines, ortryptophans. Transmembrane regions are described in, for example,http://pfam.wustl.edu/cgi-bin/getdesc?name=7tm-1, and Zagotta W. N. etal, (1996) Annual Rev. Neuronsci. 19: 235-63, the contents of which areincorporated herein by reference.

In a preferred embodiment, a 25934 polypeptide or protein has at leastone transmembrane region or a region which includes at least 20, 22, 24,25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 amino acid residues and has atleast about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a“transmembrane region,” e.g., at least one transmembrane region of human25934 (e.g., amino acid residues 50-93 or 194-235 of SEQ ID NO:2).

In one embodiment, a 25934 protein includes at least one cytoplasmicdomain. When located at the N-terminal domain the cytoplasmic domain isreferred to herein as an “N-terminal cytoplasmic domain”. As usedherein, an “N-terminal cytoplasmic domain” includes an amino acidsequence having about 1-200, preferably about 10-100, preferably about20-90, more preferably about 30-80, more preferably about 35-70, morepreferably about 40-60, or even more preferably about 45-55 amino acidresidues in length and is located in the cytoplasm of a cell. TheC-terminal amino acid residue of a “N-terminal cytoplasmic domain” isadjacent to an N-terminal amino acid residue of a transmembrane regionin a 25934 protein. For example, an N-terminal cytoplasmic domain islocated at about amino acid residues 1-49 of SEQ ID NO:2.

In a preferred embodiment, a 25934 polypeptide or protein has at leastone N-terminal cytoplasmic domain or a region which includes at leastabout 5, preferably about 40-60, or even more preferably about 45-55amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or100% homology with an “N-terminal cytoplasmic domain,” e.g., at leastone N-terminal cytoplasmic domain of human 25934 (e.g., residues 1-49 ofSEQ ID NO:2).

In another embodiment, a 25934 protein includes a “cytoplasmic loop” inthe sequence of the protein. As used herein, a “cytoplasmic loop”includes an amino acid sequence having a length of at least about 10,preferably about 20-250, preferably about 30-150, more preferably about80-120 amino acid residues and is located within the cytoplasm of acell. Accordingly, the N-terminal amino acid residue of a “cytoplasmicloop” is adjacent to a C-terminal amino acid residue of a transmembraneregion and the C-terminal residue of a “cytoplasmic loop” is adjacent toa N-terminal amino acid residue of a transmembrane region in a 25934protein. For example, a cytoplasmic loop is found at about amino acidresidues 94-193 of SEQ ID NO:2.

In a preferred embodiment, a 25934 polypeptide or protein has acytoplasmic loop or a region which includes at least about 10,preferably about 20-250, preferably about 30-150, more preferably about80-120 amino acid residues and has at least about 60%, 70% 80% 90% 95%,99%, or 100% homology with an “cytoplasmic loop,” e.g., the cytoplasmicloop of human 25934 (e.g., residues 94-193 of SEQ ID NO:2).

In another embodiment, a 25934 protein includes a “C-terminalcytoplasmic domain”, also referred to herein as a C-terminal cytoplasmictail, in the sequence of the protein. As used herein, a “C-terminalcytoplasmic domain” includes an amino acid sequence having a length ofat least about 30, preferably about 50-150, preferably about 60-200,more preferably about 80-110 amino acid residues and is located withinthe cytoplasm of a cell. Accordingly, the N-terminal amino acid residueof a “C-terminal cytoplasmic domain” is adjacent to a C-terminal aminoacid residue of a transmembrane region in a 25934 protein. For example,a C-terminal cytoplasmic domain is found at about amino acid residues236-330 of SEQ ID NO:2.

In a preferred embodiment, a 25934 polypeptide or protein has aC-terminal cytoplasmic domain or a region which includes at least about30, preferably about 50-150, preferably about 60-200, more preferablyabout 80-110 amino acid residues and has at least about 60%, 70% 80% 90%95%, 99%, or 100% homology with an “C-terminal cytoplasmic domain,”e.g., the C-terminal cytoplasmic domain of human 25934 (e.g., residues236-330 of SEQ ID NO:2).

As the 25934 polypeptides of the invention may modulate 25934-mediatedactivities, they may be useful as of for developing novel diagnostic andtherapeutic agents for 25934-mediated or related disorders, as describedbelow.

As used herein, a “25934 activity”, “biological activity of 25934” or“functional activity of 25934”, refers to an activity exerted by a 25934protein, polypeptide or nucleic acid molecule on e.g., a25934-responsive cell or on a 25934 substrate, e.g., a proteinsubstrate, as determined in vivo or in vitro. In one embodiment, a 25934activity is a direct activity, such as an association with a 25934target molecule. A “target molecule” or “binding partner” is a moleculewith which a 25934 protein binds or interacts in nature. In an exemplaryembodiment, the binding partner is a fatty acid, e.g., myristic,palmitic or stearic acid. The 25934 proteins of the present inventioncan have one or more of the following activities: (1) catalyzing theformation of a double bond, preferably, at positions up to 9 carbonsfrom the carboxyl end of a molecule, e.g., a fatty acid, such as apolyunsaturated fatty acid; (2) modulating the synthesis ofmonounsaturated fatty acids, e.g., modulating the synthesis of a fattyacid synthesized in an animal, e.g., oleic acid, palmitoyl- andstearoyl-CoA; (3) modulating the desaturation of a fatty acid, e.g., apolyunsaturated fatty acids; (4) modulating cellular lipid composition,e.g., modulating the ratio of saturated and unsaturated fatty acids; (5)modulating the energy state of adipocytes; (6) modulating membranefluidity; (7) modulating lipid storage; (8) modulating proliferationand/or differentiation; (9) modulating lipoprotein (e.g., LDL)composition and/or concentration; (10) regulating triglyceridesynthesis; (11) altering the HDL/LDL ration; or (12) modulating fattyacid metabolism.

Based on the above-described sequence similarities, the 25934 moleculesof the present invention are predicted to have similar biologicalactivities as other desaturase family members, and in particular,stearoyl CoA desaturases (SCD). For example, the 25934 polypeptide or adomain therein, e.g., desaturase domain, may function to catalyze theconversion of a single bond between two carbon atoms (C—C) to a doublebond (C═C) in a fatty acid chain. This modification is expected to occurat the n9 position of the fatty acid. Desaturases are predicted tocontribute to an unfavorable LDL content state, e.g., by increasingLDL-oleate, which is atherogenic (Rudel, L L. et al. (1997) J. ClinInvest. 1:100(1):74-83) as well as by playing a role in triglyceridemetabolism and/or biosynthesis. As shown in FIG. 7, Niacin treatment inthe marmoset model results in significant repression of 25934 in theliver. Niacin has been shown to alter the composition of LDL and HDL toa favorable state, to cause a significant reduction in triglycerides,and to increase HDL concentration (Goldberg, A. (2000) Am. J. Cardiol.85(9):1100-5. Moreover, a mouse deficient for SCD exhibits significantreduction in triglycerides (Miyazaki, M. et al. (2000) J. Biol. Chem.275(39):30132-8). Accordingly, the 25934 molecules can act as noveldiagnostic targets and therapeutic agents for controlling disordersassociated with abnormal or aberrant desaturase activity and/ortriglyceride levels. In particular, it is predicted that targeting theinhibition of 25934 nucleic acids and polypeptides will results in thefavorable modification, and possible reduction, of LDL content and/orreduction of triglycerides. Thus, the 25934 molecules can act as noveltargets for treating and/or diagnosing fatty acid metabolic disorders(e.g., desaturation of fatty acids) such as obesity and/or diabetes andmore generally, cardiovascular disorders.

Preferred examples of cardiovascular disorders or diseases include e.g.,atherosclerosis, thrombosis, heart failure, ischemic heart disease,angina pectoris, myocardial infarction, sudden cardiac death,hypertensive heart disease; non-coronary vessel disease, such asarteriolosclerosis, small vessel disease, nephropathy,hypertriglyceridemia, hypercholesterolemia, hyperlipidemia, asthma,hypertension, emphysema and chronic pulmonary disease; or acardiovascular condition associated with interventional procedures(“procedural vascular trauma”), such as restenosis followingangioplasty, placement of a shunt, stet, stent, synthetic or naturalexcision grafts, indwelling catheter, valve or other implantabledevices.

The term “cardiovascular disorders” or “disease” includes heartdisorders, as well as disorders of the blood vessels of the circulationsystem caused by, e.g., abnormally high concentrations of lipids in theblood vessels.

Disorders involving the heart, include but are not limited to, heartfailure, including but not limited to, cardiac hypertrophy, left-sidedheart failure, and right-sided heart failure; ischemic heart disease,including but not limited to angina pectoris, myocardial infarction,chronic ischemic heart disease, and sudden cardiac death; hypertensiveheart disease, including but not limited to, systemic (left-sided)hypertensive heart disease and pulmonary (right-sided) hypertensiveheart disease; valvular heart disease, including but not limited to,valvular degeneration caused by calcification, such as calcific aorticstenosis, calcification of a congenitally bicuspid aortic valve, andmitral annular calcification, and myxomatous degeneration of the mitralvalve (mitral valve prolapse), rheumatic fever and rheumatic heartdisease, infective endocarditis, and noninfected vegetations, such asnonbacterial thrombotic endocarditis and endocarditis of systemic lupuserythematosus (Libman-Sacks disease), carcinoid heart disease, andcomplications of artificial valves; myocardial disease, including butnot limited to dilated cardiomyopathy, hypertrophic cardiomyopathy,restrictive cardiomyopathy, and myocarditis; pericardial disease,including but not limited to, pericardial effusion and hemopericardiumand pericarditis, including acute pericarditis and healed pericarditis,and rheumatoid heart disease; neoplastic heart disease, including butnot limited to, primary cardiac tumors, such as myxoma, lipoma,papillary fibroelastoma, rhabdomyoma, and sarcoma, and cardiac effectsof noncardiac neoplasms; congenital heart disease, including but notlimited to, left-to-right shunts--late cyanosis, such as atrial septaldefect, ventricular septal defect, patent ductus arteriosus, andatrioventricular septal defect, right-to-left shunts—early cyanosis,such as tetralogy of fallot, transposition of great arteries, truncusarteriosus, tricuspid atresia, and total anomalous pulmonary venousconnection, obstructive congenital anomalies, such as coarctation ofaorta, pulmonary stenosis and atresia, and aortic stenosis and atresia,and disorders involving cardiac transplantation.

Disorders involving blood vessels include, but are not limited to,responses of vascular cell walls to injury, such as endothelialdysfunction and endothelial activation and intimal thickening; vasculardiseases including, but not limited to, congenital anomalies, such asarteriovenous fistula, atherosclerosis, and hypertensive vasculardisease, such as hypertension; inflammatory disease—the vasculitides,such as giant cell (temporal) arteritis, Takayasu arteritis,polyarteritis nodosa (classic), Kawasaki syndrome (mucocutaneous lymphnode syndrome), microscopic polyanglitis (microscopic polyarteritis,hypersensitivity or leukocytoclastic anglitis), Wegener granulomatosis,thromboanglitis obliterans (Buerger disease), vasculitis associated withother disorders, and infectious arteritis; Raynaud disease; aneurysmsand dissection, such as abdominal aortic aneurysms, syphilitic (luetic)aneurysms, and aortic dissection (dissecting hematoma); disorders ofveins and lymphatics, such as varicose veins, thrombophlebitis andphlebothrombosis, obstruction of superior vena cava (superior vena cavasyndrome), obstruction of inferior vena cava (inferior vena cavasyndrome), and lymphangitis and lymphedema; tumors, including benigntumors and tumor-like conditions, such as hemangioma, lymphangioma,glomus tumor (glomangioma), vascular ectasias, and bacillaryangiomatosis, and intermediate-grade (borderline low-grade malignant)tumors, such as Kaposi sarcoma and hemangloendothelioma, and malignanttumors, such as angiosarcoma and hemangiopericytoma; and pathology oftherapeutic interventions in vascular disease, such as balloonangioplasty and related techniques and vascular replacement, such ascoronary artery bypass graft surgery.

As used herein, the term “atherosclerosis” is intended to have itsclinical meaning. This term refers to a cardiovascular conditionoccurring as a result of narrowing down of the arterial walls. Thenarrowing is due to the formation of plaques (raised patches) or streaksin the inner lining of the arteries. These plaques consist of foam cellsof low-density lipoproteins, oxidized-LDL, decaying muscle cells,fibrous tissue, clumps of blood platelets, cholesterol, and sometimescalcium. They tend to form in regions of turbulent blood flow and arefound most often in people with high concentrations of cholesterol inthe bloodstream. The number and thickness of plaques increase with age,causing loss of the smooth lining of the blood vessels and encouragingthe formation of thrombi (blood clots). Sometimes fragments of thrombibreak off and form emboli, which travel through the bloodstream andblock smaller vessels. The blood supply is restricted to the heart,eventually forming a blood clot leading to death. The major causes ofatherosclerosis are hypercholesterolemia (and low HDL),hypoalphoproteinemia, and hyperlipidemia marked by high circulatingcholesterol and high lipids like LDL-cholesterol and triglycerides inthe blood. These lipids are deposited in the arterial walls, obstructingthe blood flow and forming atherosclerotic plaques leading to death.

As used herein the term “hypercholesterolemia” is a condition withelevated levels of circulating total cholesterol, LDL-cholesterol andVLDL-cholesterol as per the guidelines of the Expert Panel Report of theNational Cholesterol Educational Program (NCEP) of Detection, Evaluationof Treatment of high cholesterol in adults (see, Arch. Int. Med. (1988)148, 36-39).

As used herein the term “hyperlipidemia” or “hyperlipemia” is acondition where the blood lipid parameters are elevated in the blood.This condition manifests an abnormally high concentration of fats. Thelipid fractions in the circulating blood are, total cholesterol, lowdensity lipoproteins, very low density lipoproteins and triglycerides.

As used herein the term “lipoprotein” such as VLDL, LDL and HDL, refersto a group of proteins found in the serum, plasma and lymph and areimportant for lipid transport. The chemical composition of eachlipoprotein differs in that the HDL has a higher proportion of proteinversus lipid, whereas the VLDL has a lower proportion of protein versuslipid.

As used herein, the term “triglyceride” means a lipid or neutral fatconsisting of glycerol combined with three fatty acid molecules.

As used herein the term “xanthomatosis” is a disease evidenced by ayellowish swelling or plaques in the skin resulting from deposits offat. The presence of xanthomas are usually accompanied by raised bloodcholesterol levels.

As used herein the term “apolipoprotein B” or “apoprotein B” or “Apo B”refers to the protein component of the LDL cholesterol transportproteins. Cholesterol synthesized de novo is transported from the liverand intestine to peripheral tissues in the form of lipoproteins. Most ofthe apolipoprotein B is secreted into the circulatory system as VLDL.

As used herein the term “apolipoprotein A” or “apoprotein A” or “Apo A”refers to the protein component of the HDL cholesterol transportproteins.

“Procedural vascular trauma” includes the effects ofsurgical/medical-mechanical interventions into mammalian vasculature,but does not include vascular trauma due to the organic vascularpathologies listed hereinabove, or to unintended traumas, such as due toan accident. Thus, procedural vascular traumas within the scope of thepresent treatment method include (1) organ grafting or transplantation,such as transplantation and grafting of heart, kidney, liver and thelike, e.g., involving vessel anastomosis; (2) vascular surgery, such ascoronary bypass surgery, biopsy, heart valve replacement, atheroectomy,thrombectomy, and the like; (3) transcatheter vascular therapies (TVT)including angioplasty, e.g., laser angioplasty and PTCA proceduresdiscussed hereinbelow, employing balloon catheters, or indwellingcatheters; (4) vascular grafting using natural or synthetic materials,such as in saphenous vein coronary bypass grafts, dacron and venousgrafts used for peripheral arterial reconstruction, etc.; (5) placementof a mechanical shunt, such as a PTFE hemodialysis shunt used forarteriovenous communications; and (6) placement of an intravascularstent, which may be metallic, plastic or a biodegradable polymer. SeeU.S. patent application Ser. No. 08/389,712, filed Feb. 15, 1995, whichis incorporated by reference herein. For a general discussion ofimplantable devices and biomaterials from which they can be formed, seeH. Kambic et al., “Biomaterials in Artificial Organs”, Chem. Eng. News,30 (Apr. 14, 1986), the disclosure of which is incorporated by referenceherein.

Small vessel disease includes, but is not limited to, vascularinsufficiency in the limbs, peripheral neuropathy and retinopathy, e.g.,diabetic retinopathy.

In some embodiments, the therapeutic and prophylactic uses of thecompositions of the invention, further include the administration ofcholesterol lowering agents as a combination drug therapies. The term“combination therapy” as used herein refers to the administration to asubject (concurrently or sequentially) of two or more cholesterollowering agents. Current combination therapy therapies usingcombinations of niacin and statins are being used with positive resultsto treat hyperlipidemia (Guyton, J R. (1999) Curr Cardiol Rep.1(3):244-250; Otto, C. et al. (1999) Internist (Berl) 40(12):1338-45).Other useful drug combinations include those derived by addition of fishoil, bile acid binding resins, or stanol esters, as well as nonstatincombinations susn as niacin-resin or fibrate-niacin (Guyton, J R. (1999)supra). For examples of dosages and administration schedules of thecholesterol lowering agents, the teachings of Guyton, J R. (1999) supra,Otto, C. et al. (1999) supra, Guyton, J R et al. (1998) Am J Cardiol82(12A):82U-86U; Guyton, J R et al. (1998) Am J Cardiol. 82(6):737-43;Vega, G L et al. (1998) Am J. Cardiol. 81(4A):36B-42B; Schectman, G.(1996) Ann Intern Med. 125(12):990-1000; Nakamura, H. et al. (1993)Nippon Rinsho 51(8):2101-7; Goldberg, A. et al. (2000) Am J Cardiol85(9):1100-5; Morgan, J M et al. (1996) J Cardiovasc. Pharmac. Ther.1(3):195-202; Stein, E A et al. (1996) J Cardiovasc Pharmacol Ther1(2):107-116; and Goldberg, A C (1998) Am J Cardiol 82(12A):35U-41U, areexpressly incorporated by reference.

As used herein, “cholesterol lowering agents” include agents which areuseful for lowering serum cholesterol such as for example bile acidsequestering resins (e.g. colestipol hydrochloride or cholestyramine),fish oil, stanol esters, an ApoAII-lowering agent, a VLDL loweringagent, an ApoAI-stimulating agent, fibric acid derivatives (e.g.clofibrate, fenofibrate, or gemfibrozil), thiazolidenediones (e.g.troglitazone), or HMG-CoA reductase inhibitors (e.g. statins, such asfluvastatin sodium, lovastatin, pravastatin sodium, or simvastatin), aswell as nicotinic acid, niacin, or probucol.

“VLDL-lowering agent” includes an agent which decreases the hepaticsynthesis of triglyceride-rich lipoproteins or increases the catabolismof triglyceride-rich lipoproteins, e.g., fibrates such as gemfibrozil,or the statins, increases the expression of the apoE-mediated clearancepathway, or improves insulin sensitivity in diabetics, e.g., thethiazolidene diones.

The 25934 molecules can also be used to treat, diagnose or prevent lipiddisorders. Examples of lipid disorders include those disorders whichaffect fatty acid metabolism. Fatty acids are synthesized fromacetyl-CoA, which is derived from carbohydrate, protein and othernon-lipid sources, and the pathway produces saturated fatty acids,predominantly palmitic acid (10:0). In mammals, the fatty acids may beelongated and desaturated. Desaturation is catalyzed by desaturaseswhich function by inserting one or more double bonds at positions up to9 carbons from the carboxyl end of a fatty acid molecule.

The degree of fatty acid desaturation in cell membrane lipids determinesmembrane fluidity. The activity of the desaturase enzyme is critical formaintaining the ratio of saturated and unsaturated fatty acids in cellmembranes. Alterations in this ratio can, e.g., alter the physicalproperties of membranes. Moreover, alterations in the ratio of fattyacids have been implicated in a range of diseases including diabetes,obesity, hypertension, cancer, developmental disorders, immune disordersand neurological and the above-described heart diseases. For example,tumor tissue and virus-transformed cells have a higher content ofunsaturated fatty acids, especially oleic acid. Such shifts increase themetabolic rates of many lipid-dependent enzymes and are associated witha higher capacity for cell division.

As the 25934 mRNA is found in the brain, ovary, kidney and liver, themolecules of the invention can be used to develop novel agents orcompounds to treat, prevent and/or diagnose disorders involving aberrantactivities of those cells (FIGS. 4-6). For example, the molecules of theinvention can be used to treat, present and/or diagnose neurological,reproductive (ovarian), renal and hepatic disorders, as described below.

The 25934 protein, fragments thereof, and derivatives and other variantsof the sequence in SEQ ID NO:2 thereof are collectively referred to as“polypeptides or proteins of the invention” or “25934 polypeptides orproteins”. Nucleic acid molecules encoding such polypeptides or proteinsare collectively referred to as “nucleic acids of the invention” or“25934 nucleic acids.” 25934 molecules refer to 25934 nucleic acids,polypeptides, and antibodies.

As used herein, the term “nucleic acid molecule” includes DNA molecules(e.g., a cDNA or genomic DNA) and RNA molecules (e.g., an mRNA) andanalogs of the DNA or RNA generated, e.g., by the use of nucleotideanalogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

The term “isolated or purified nucleic acid molecule” includes nucleicacid molecules which are separated from other nucleic acid moleculeswhich are present in the natural source of the nucleic acid. Forexample, with regards to genomic DNA, the term “isolated” includesnucleic acid molecules which are separated from the chromosome withwhich the genomic DNA is naturally associated. Preferably, an “isolated”nucleic acid is free of sequences which naturally flank the nucleic acid(i.e., sequences located at the 5′ and/or 3′ ends of the nucleic acid)in the genomic DNA of the organism from which the nucleic acid isderived. For example, in various embodiments, the isolated nucleic acidmolecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5kb or 0.1 kb of 5′ and/or 3′ nucleotide sequences which naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized.

As used herein, the term “hybridizes under stringent conditions”describes conditions for hybridization and washing. Stringent conditionsare known to those skilled in the art and can be found in CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. Aqueous and nonaqueous methods are described in thatreference and either can be used. A preferred, example of stringenthybridization conditions are hybridization in 6×sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50° C. Another example of stringent hybridizationconditions are hybridization in 6×sodium chloride/sodium citrate (SSC)at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at55° C. A further example of stringent hybridization conditions arehybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C.,followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.Preferably, stringent hybridization conditions are hybridization in6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by oneor more washes in 0.2×SSC, 0.1% SDS at 65° C. Particularly preferredstringency conditions (and the conditions that should be used if thepractitioner is uncertain about what conditions should be applied todetermine if a molecule is within the hybridization limits of the claim)are 0.5M Sodium Phosphate, 7% SDS at 65° C., followed by one or morewashes at 0.2×SSC, 1% SDS at 65° C. Preferably, an isolated nucleic acidmolecule of the invention that hybridizes under stringent conditions tothe sequence of SEQ ID NO:1 or 3, corresponds to a naturally-occurringnucleic acid molecule.

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g., encodes a natural protein).

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules which include an open reading frame encoding a 25934protein, preferably a mammalian 25934 protein, and can further includenon-coding regulatory sequences, and introns.

An “isolated” or “purified” polypeptide or protein is substantially freeof cellular material or other contaminating proteins from the cell ortissue source from which the protein is derived, or substantially freefrom chemical precursors or other chemicals when chemically synthesized.In one embodiment, the language “substantially free” means preparationof 25934 protein having less than about 30%, 20%, 10% and morepreferably 5% (by dry weight), of non-25934 protein (also referred toherein as a “contaminating protein”), or of chemical precursors ornon-25934 chemicals. When the 25934 protein or biologically activeportion thereof is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 20%, more preferably less than about 10%, and mostpreferably less than about 5% of the volume of the protein preparation.The invention includes isolated or purified preparations of at least0.01, 0.1, 1.0, and 10 milligrams in dry weight.

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of 25934 (e.g., the sequence of SEQ ID NO:1or 3, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 2167) without abolishing or morepreferably, without substantially altering a biological activity,whereas an “essential” amino acid residue results in such a change. Forexample, amino acid residues that are conserved among the polypeptidesof the present invention, e.g., those present in the desaturase domain,are predicted to be particularly unamenable to alteration.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a 25934 protein ispreferably replaced with another amino acid residue from the same sidechain family. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a 25934 coding sequence, suchas by saturation mutagenesis, and the resultant mutants can be screenedfor 25934 biological activity to identify mutants that retain activity.Following mutagenesis of SEQ ID NO:1 or 3, or the nucleotide sequence ofthe DNA insert of the plasmid deposited with ATCC as Accession Number2167, the encoded protein can be expressed recombinantly and theactivity of the protein can be determined.

As used herein, a “biologically active portion” of a 25934 proteinincludes a fragment of a 25934 protein which participates in aninteraction between a 25934 molecule and a non-25934 molecule.Biologically active portions of a 25934 protein include peptidescomprising amino acid sequences sufficiently homologous to or derivedfrom the amino acid sequence of the 25934 protein, e.g., the amino acidsequence shown in SEQ ID NO:2, which include less amino acids than thefull length 25934 proteins, and exhibit at least one activity of a 25934protein. Typically, biologically active portions comprise a domain ormotif with at least one activity of the 25934 protein, e.g., 1)catalyzing the formation of a double bond at positions up to 9 carbonsfrom the carboxyl end of a molecule; (2) modulating the synthesis ofmonounsaturated fatty acids; (3) modulating the desaturation of a fattyacid, e.g., a polyunsaturated fatty acids; (4) modulating cellular lipidcomposition; (5) modulating the energy state of adipocytes; (6)modulating membrane fluidity; (7) modulating lipid storage; (8)modulating cell proliferation and/or differentiation; (9) modulatinglipoprotein (e.g., LDL) composition and/or concentration; (10)regulating triglyceride synthesis; (11) altering the HDL/LDL ration; or(12) modulating fatty acid metabolism. A biologically active portion ofa 25934 protein can be a polypeptide which is, for example, 50, 100, 200or more amino acids in length. Biologically active portions of a 25934protein can be used as targets for developing agents which modulate a25934 mediated activity, e.g., a 25934 mediated activity as describedabove.

Particular 25934 polypeptides of the present invention have an aminoacid sequence sufficiently identical to the amino acid sequence of SEQID NO:2. The term “sufficiently identical” or “substantially identical”is used herein to refer to a first amino acid or nucleotide sequencethat contains a sufficient or minimum number of identical or equivalent(e.g., with a similar side chain) amino acid residues or nucleotides toa second amino acid or nucleotide sequence such that the first andsecond amino acid or nucleotide sequences have a common structuraldomain or common functional activity. For example, amino acid ornucleotide sequences that contain a common structural domain having atleast about 60-65% identity, likely 66-70%, 75% identity, more likely85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity aredefined herein as sufficiently or substantially identical.

Calculations of homology or sequence identity between sequences (theterms are used interchangeably herein) are performed as follows.

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, 90%, 100% of the length of thereference sequence (e.g., when aligning a second sequence to the 25934amino acid sequence of SEQ ID NO:2 having 20 amino acid residues, atleast 50, preferably at least 100, more preferably at least 200, evenmore preferably at least 300, and even more preferably at least 320, or330 amino acid residues are aligned). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Aparticularly preferred set of parameters (and the one that should beused if the practitioner is uncertain about what parameters should beapplied to determine if the molecule is within the sequence identitylimits of a claim) is using Blossum 62 scoring matrix with a gap openpenalty of 12, a gap extend penalty of 4, and a frameshift gap penaltyof 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of E. Meyers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to 25934 nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to 25934 protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See http://www.ncbi.nlm.nih.gov.

“Misexpression or aberrant expression”, as used herein, refers to anon-wild type pattern of gene expression, at the RNA or protein level.It includes: expression at non-wild type levels, i.e., over or underexpression; a pattern of expression that differs from wild type in termsof the time or stage at which the gene is expressed, e.g., increased ordecreased expression (as compared with wild type) at a predetermineddevelopmental period or stage; a pattern of expression that differs fromwild type in terms of decreased expression (as compared with wild type)in a predetermined cell type or tissue type; a pattern of expressionthat differs from wild type in terms of the splicing size, amino acidsequence, post-transitional modification, or biological activity of theexpressed polypeptide; a pattern of expression that differs from wildtype in terms of the effect of an environmental stimulus orextracellular stimulus on expression of the gene, e.g., a pattern ofincreased or decreased expression (as compared with wild type) in thepresence of an increase or decrease in the strength of the stimulus.

“Subject”, as used herein, can refer to a mammal, e.g., a human, or toan experimental or animal or disease model. The subject can also be anon-human animal, e.g., a horse, cow, goat, or other domestic animal.

A “purified preparation of cells”, as used herein, refers to, in thecase of plant or animal cells, an in vitro preparation of cells and notan entire intact plant or animal. In the case of cultured cells ormicrobial cells, it consists of a preparation of at least 10% and morepreferably 50% of the subject cells.

Various aspects of the invention are described in further detail below.

Isolated Nucleic Acid Molecules

In one aspect, the invention provides, an isolated or purified, nucleicacid molecule that encodes a 25934 polypeptide described herein, e.g., afull length 25934 protein or a fragment thereof, e.g., a biologicallyactive portion of 25934 protein. Also included is a nucleic acidfragment suitable for use as a hybridization probe, which can be used,e.g., to a identify nucleic acid molecule encoding a polypeptide of theinvention, 25934 mRNA, and fragments suitable for use as primers, e.g.,PCR primers for the amplification or mutation of nucleic acid molecules.

In one embodiment, an isolated nucleic acid molecule of the inventionincludes the nucleotide sequence shown in SEQ ID NO:1, or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number 2167, or a portion of any of these nucleotidesequences. In one embodiment, the nucleic acid molecule includessequences encoding the human 25934 protein (i.e., “the coding region”,from nucleotides 403-1392 of SEQ ID NO:1), as well as 5′ untranslatedsequence (nucleotides 1-402 of SEQ ID NO:1) and a 3′ untranslatedsequence (nucleotides 1393-1512). Alternatively, the nucleic acidmolecule can include only the coding region of SEQ ID NO:1 (e.g.,nucleotides 1-990, corresponding to SEQ ID NO:3) and, e.g., no flankingsequences which normally accompany the subject sequence.

In another embodiment, an isolated nucleic acid molecule of theinvention includes a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO:1 or 3, or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number 2167, or a portion of any of these nucleotidesequences. In other embodiments, the nucleic acid molecule of theinvention is sufficiently complementary to the nucleotide sequence shownin SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC as Accession Number 2167 such that it canhybridize to the nucleotide sequence shown in SEQ ID NO:1 or 3, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 2167, thereby forming a stable duplex.

In one embodiment, an isolated nucleic acid molecule of the presentinvention includes a nucleotide sequence which is at least about: 60%,65%, 66-69%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more homologous to the entire length of the nucleotidesequence shown in SEQ ID NO:1 or 3, or the entire length of thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 2167, or a portion, preferably of the same length,of any of these nucleotide sequences.

25934 Nucleic Acid Fragments

A nucleic acid molecule of the invention can include only a portion ofthe nucleic acid sequence of SEQ ID NO:1 or 3, or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number 2167. For example, such a nucleic acid molecule caninclude a fragment which can be used as a probe or primer or a fragmentencoding a portion of a 25934 protein, e.g., an immunogenic orbiologically active portion of a 25934 protein. A fragment can comprisenucleotides 555 to 1287 of SEQ ID NO:1, which encodes a desaturasedomain of human 25934. The nucleotide sequence determined from thecloning of the 25934 gene allows for the generation of probes andprimers designed for use in identifying and/or cloning other 25934family members, or fragments thereof, as well as 25934 homologues, orfragments thereof, from other species.

In another embodiment, a nucleic acid includes a nucleotide sequencethat includes part, or all, of the coding region and extends into either(or both) the 5′ or 3′ noncoding region. Other embodiments include afragment which includes a nucleotide sequence encoding an amino acidfragment described herein. Nucleic acid fragments can encode a specificdomain or site described herein or fragments thereof, particularyfragments thereof which are at least 50, 90, 120, 150, 200, or 244 aminoacids in length. Fragments also include nucleic acid sequencescorresponding to specific amino acid sequences described above orfragments thereof. Nucleic acid fragments should not to be construed asencompassing those fragments that may have been disclosed prior to theinvention.

A nucleic acid fragment can include a sequence corresponding to adomain, region, or functional site described herein. A nucleic acidfragment can also include one or more domain, region, or functional sitedescribed herein. Thus, for example, the fragment can include adesaturase domain and a protein kinase C phosphorylation site.

25934 probes and primers are provided. Typically a probe/primer is anisolated or purified oligonucleotide. The oligonucleotide typicallyincludes a region of nucleotide sequence that hybridizes under stringentconditions to at least about 7, 12 or 15, preferably about 20 or 25,more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutivenucleotides of a sense or antisense sequence of SEQ ID NO:1 or 3, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 2167, or of a naturally occurring allelic variant ormutant of SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insertof the plasmid deposited with ATCC as Accession Number 2167.

In a preferred embodiment the nucleic acid is a probe which is at least5 or 10, and less than 200, more preferably less than 100, or less than50, base pairs in length. It should be identical, or differ by 1, orless than in 5 or 10 bases, from a sequence disclosed herein. Ifalignment is needed for this comparison the sequences should be alignedfor maximum homology. “Looped” out sequences from deletions orinsertions, or mismatches, are considered differences.

A probe or primer can be derived from the sense or anti-sense strand ofa nucleic acid which encodes a desaturase domain: amino acids 51 to 295of SEQ ID NO:2.

In another embodiment a set of primers is provided, e.g., primerssuitable for use in a PCR, which can be used to amplify a selectedregion of a 25934 sequence. The primers should be at least 5, 10, or 50base pairs in length and less than 100, or less than 200, base pairs inlength. The primers should be identical, or differs by one base from asequence disclosed herein or from a naturally occurring variant. E.g.,primers suitable for amplifying a domain or region described herein,e.g., a desaturase domain which occurs at amino acids 51 to 295 of SEQID NO:2, are provided.

A nucleic acid fragment can encode an epitope bearing region of apolypeptide described herein.

A nucleic acid fragment encoding a “biologically active portion of a25934 polypeptide” can be prepared by isolating a portion of thenucleotide sequence of SEQ ID NO:1 or 3, or the nucleotide sequence ofthe DNA insert of the plasmid deposited with ATCC as Accession Number2167, which encodes a polypeptide having a 25934 biological activity(e.g., the biological activities of the 25934 proteins are describedherein), expressing the encoded portion of the 25934 protein (e.g., byrecombinant expression in vitro) and assessing the activity of theencoded portion of the 25934 protein. For example, a nucleic acidfragment encoding a biologically active portion of 25934 includes adesaturase domain, e.g., amino acid residues 51 to 295 of SEQ ID NO:2. Anucleic acid fragment encoding a biologically active portion of a 25934polypeptide, may comprise a nucleotide sequence which is greater 350 ormore nucleotides in length (e.g., greater than about 400 nucleotides inlength).

In preferred embodiment, the fragment is at least 604, 650, 700, 750,800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500 nucleotides inlength, or more nucleotides in length and hybridizes under stringenthybridization conditions to a nucleic acid molecule of SEQ ID NO:1, orSEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 2167.

In a preferred embodiment, a nucleic acid fragment includes a nucleotidesequence comprising nucleotides SEQ ID NO:1 or SEQ ID NO:3, or a portionthereof, wherein each portion is about 604 or longer nucleotides andhybridizes under stringent hybridization conditions to a nucleic acidmolecule of SEQ ID NO:1, or SEQ ID NO:3, or the nucleotide sequence ofthe DNA insert of the plasmid deposited with ATCC as Accession Number2167. In other embodiments, a nucleic acid fragment includes anucleotide sequence of about 466 or more nucleotides in length whichcomprises nucleotides 1-709 of SEQ ID NO:1; or about 604 or morenucleotides in length which comprises nucleotides 710-1669 of SEQ IDNO:1.

In a preferred embodiment, a nucleic acid fragment has a nucleotidesequence other than (e.g., differs by at least one, two, three, five,ten or more nucleotides from) the nucleotide sequence of sequence of AI401562, AI 942480, AW 131469, BE 515130, W 28157, BE 244746, AI 815730,or AI 816228.

25934 Nucleic Acid Variants

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:1 or 3, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 2167. Such differences can be due to degeneracy ofthe genetic code (and result in a nucleic acid which encodes the same25934 proteins as those encoded by the nucleotide sequence disclosedherein. In another embodiment, an isolated nucleic acid molecule of theinvention has a nucleotide sequence encoding a protein having an aminoacid sequence which differs, by at least 1, but less than 5, 10, 20, 50,or 100 amino acid residues that shown in SEQ ID NO:2. If alignment isneeded for this comparison the sequences should be aligned for maximumhomology. “Looped” out sequences from deletions or insertions, ormismatches, are considered differences.

Nucleic acids of the inventor can be chosen for having codons, which arepreferred, or non preferred, for a particular expression system. E.g.,the nucleic acid can be one in which at least one colon, at preferablyat least 10%, or 20% of the codons has been altered such that thesequence is optimized for expression in e. coli, yeast, human, insect,or CHO cells.

Nucleic acid variants can be naturally occurring, such as allelicvariants (same locus), homologs (different locus), and orthologs(different organism) or can be non naturally occurring. Non-naturallyoccurring variants can be made by mutagenesis techniques, includingthose applied to polynucleotides, cells, or organisms. The variants cancontain nucleotide substitutions, deletions, inversions and insertions.Variation can occur in either or both the coding and non-coding regions.The variations can produce both conservative and non-conservative aminoacid substitutions (as compared in the encoded product).

In a preferred embodiment, the nucleic acid differs from that of SEQ IDNO:1 or 3, or the sequence in ATCC Accession Number 2167, e.g., asfollows: by at least one but less than 10, 20, 30, or 40 nucleotides; atleast one but less than 1%, 5%, 10% or 20% of the in the subject nucleicacid. If necessary for this analysis the sequences should be aligned formaximum homology. “Looped” out sequences from deletions or insertions,or mismatches, are considered differences.

Orthologs, homologs, and allelic variants can be identified usingmethods known in the art. These variants comprise a nucleotide sequenceencoding a polypeptide that is 50%, at least about 55%, typically atleast about 70-75%, more typically at least about 80-85%, and mosttypically at least about 90-95% or more identical to the nucleotidesequence shown in SEQ ID NO:2 or a fragment of this sequence. Nucleicacid molecules corresponding to orthologs, homologs, and allelicvariants of the 25934 cDNAs of the invention can further be isolated bymapping to the same chromosome or locus as the 25934 gene.

Preferred variants include those that are correlated with (1) catalyzingthe formation of a double bond, preferably, at positions up to 9 carbonsfrom the carboxyl end of a molecule, e.g., a fatty acid, such as apolyunsaturated fatty acid; (2) modulating the synthesis ofmonounsaturated fatty acids, e.g., modulating the synthesis of a fattyacid synthesized in an animal, e.g., oleic acid, palmitoyl- andstearoyl-CoA; (3) modulating the desaturation of a fatty acid, e.g., apolyunsaturated fatty acids; (4) modulating cellular lipid composition,e.g., modulating the ratio of saturated and unsaturated fatty acids; (5)modulating the energy state of adipocytes; (6) modulating membranefluidity; (7) modulating lipid storage; (8) modulating proliferationand/or differentiation; (9) modulating lipoprotein (e.g., LDL)composition and/or concentration; (10) regulating triglyceridesynthesis; (11) altering the HDL/LDL ration; (12) modulating fatty acidmetabolism; or (13) modulating the development of atheroscleroticlesions in disease models.

Allelic variants of 25934, e.g., human 25934, include both functionaland non-functional proteins. Functional allelic variants are naturallyoccurring amino acid sequence variants of the 25934 protein within apopulation that maintain the ability to (1) catalyzing the formation ofa double bond, preferably, at positions up to 9 carbons from thecarboxyl end of a molecule, e.g., a fatty acid, such as apolyunsaturated fatty acid; (2) modulating the synthesis ofmonounsaturated fatty acids, e.g., modulating the synthesis of a fattyacid synthesized in an animal, e.g., oleic acid, palmitoyl- andstearoyl-CoA; (3) modulating the desaturation of a fatty acid, e.g., apolyunsaturated fatty acids; (4) modulating cellular lipid composition,e.g., modulating the ratio of saturated and unsaturated fatty acids; (5)modulating the energy state of adipocytes; (6) modulating membranefluidity; (7) modulating lipid storage; (8) modulating proliferationand/or differentiation; (9) modulating lipoprotein (e.g., LDL)composition and/or concentration; (10) regulating triglyceridesynthesis; (11) altering the HDL/LDL ration; or (12) modulating fattyacid metabolism. Functional allelic variants will typically contain onlyconservative substitution of one or more amino acids of SEQ ID NO:2, orsubstitution, deletion or insertion of non-critical residues innon-critical regions of the protein. Non-functional allelic variants arenaturally-occurring amino acid sequence variants of the 25934, e.g.,human 25934, protein within a population that do not have the ability tomodulate a 25934-mediated activity as described herein. Non-functionalallelic variants will typically contain a non-conservative substitution,a deletion, or insertion, or premature truncation of the amino acidsequence of SEQ ID NO:2, or a substitution, insertion, or deletion incritical residues or critical regions of the protein.

Moreover, nucleic acid molecules encoding other 25934 family membersand, thus, which have a nucleotide sequence which differs from the 25934sequences of SEQ ID NO:1 or 3, or the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession Number 2167 areintended to be within the scope of the invention.

Antisense Nucleic Acid Molecules, Ribozymes and Modified 25934 NucleicAcid Molecules

In another aspect, the invention features, an isolated nucleic acidmolecule which is antisense to 25934. An “antisense” nucleic acid caninclude a nucleotide sequence which is complementary to a “sense”nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an mRNAsequence. The antisense nucleic acid can be complementary to an entire25934 coding strand, or to only a portion thereof (e.g., the codingregion of human 25934 corresponding to SEQ ID NO:3). In anotherembodiment, the antisense nucleic acid molecule is antisense to a“noncoding region” of the coding strand of a nucleotide sequenceencoding 25934 (e.g., the 5′ and 3′ untranslated regions).

An antisense nucleic acid can be designed such that it is complementaryto the entire coding region of 25934 mRNA, but more preferably is anoligonucleotide which is antisense to only a portion of the coding ornoncoding region of 25934 mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of 25934 mRNA, e.g., between the −10 and +10regions of the target gene nucleotide sequence of interest. An antisenseoligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.

An antisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (e.g., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. The antisense nucleic acid also canbe produced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject (e.g., by direct injection at a tissue site),or generated in situ such that they hybridize with or bind to cellularmRNA and/or genomic DNA encoding a 25934 protein to thereby inhibitexpression of the protein, e.g., by inhibiting transcription and/ortranslation. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For systemic administration, antisense molecules can be modified suchthat they specifically bind to receptors or antigens expressed on aselected cell surface, e.g., by linking the antisense nucleic acidmolecules to peptides or antibodies which bind to cell surface receptorsor antigens. The antisense nucleic acid molecules can also be deliveredto cells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong pol II or pol III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. A ribozyme having specificity for a 25934-encodingnucleic acid can include one or more sequences complementary to the thenucleotide sequence of a 25934 cDNA disclosed herein (i.e., SEQ ID NO:1or 3), and a sequence having known catalytic sequence responsible formRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach(1988) Nature 334:585-591). For example, a derivative of a TetrahymenaL-19 IVS RNA can be constructed in which the nucleotide sequence of theactive site is complementary to the nucleotide sequence to be cleaved ina 25934-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071;and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, 25934 mRNA canbe used to select a catalytic RNA having a specific ribonucleaseactivity from a pool of RNA molecules. See, e.g., Bartel, D. andSzostak, J. W. (1993) Science 261:1411-1418.

25934 gene expression can be inhibited by targeting nucleotide sequencescomplementary to the regulatory region of the 25934 (e.g., the 25934promoter and/or enhancers) to form triple helical structures thatprevent transcription of the 25934 gene in target cells. See generally,Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al.(1992) Ann. N.Y Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays14(12):807-15. The potential sequences that can be targeted for triplehelix formation can be increased by creating a so called “switchback”nucleic acid molecule. Switchback molecules are synthesized in analternating 5′-3′, 3′-5′ manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizeable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

The invention also provides detectably labeled oligonucleotide primerand probe molecules. Typically, such labels are chemiluminescent,fluorescent, radioactive, or colorimetric.

A 25934 nucleic acid molecule can be modified at the base moiety, sugarmoiety or phosphate backbone to improve, e.g., the stability,hybridization, or solubility of the molecule. For example, thedeoxyribose phosphate backbone of the nucleic acid molecules can bemodified to generate peptide nucleic acids (see Hyrup B. et al. (1996)Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the terms“peptide nucleic acid” or “PNA” refers to a nucleic acid mimic, e.g., aDNA mimic, in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of a PNA can allow for specifichybridization to DNA and RNA under conditions of low ionic strength. Thesynthesis of PNA oligomers can be performed using standard solid phasepeptide synthesis protocols as described in Hyrup B. et al. (1996)supra; Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.

PNAs of 25934 nucleic acid molecules can be used in therapeutic anddiagnostic applications. For example, PNAs can be used as antisense orantigene agents for sequence-specific modulation of gene expression by,for example, inducing transcription or translation arrest or inhibitingreplication. PNAs of 25934 nucleic acid molecules can also be used inthe analysis of single base pair mutations in a gene, (e.g., byPNA-directed PCR clamping); as ‘artificial restriction enzymes’ whenused in combination with other enzymes, (e.g., S1 nucleases (Hyrup B.(1996) supra)); or as probes or primers for DNA sequencing orhybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. W088/09810) or the blood-brain barrier (see, e.g., PCTPublication No. W089/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (See, e.g., Krolet al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (See,e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, (e.g., a peptide,hybridization triggered cross-linking agent, transport agent, orhybridization-triggered cleavage agent).

The invention also includes molecular beacon oligonucleotide primer andprobe molecules having at least one region which is complementary to a25934 nucleic acid of the invention, two complementary regions onehaving a fluorophore and one a quencher such that the molecular beaconis useful for quantitating the presence of the 25934 nucleic acid of theinvention in a sample. Molecular beacon nucleic acids are described, forexample, in Lizardi et al., U.S. Pat. No. 5,854,033; Nazarenko et al.,U.S. Pat. No. 5,866,336, and Livak et al., U.S. Pat. No. 5,876,930.

Isolated 25934 Polypeptides

In another aspect, the invention features, an isolated 25934 protein, orfragment, e.g., a biologically active portion, for use as immunogens orantigens to raise or test (or more generally to bind) anti-25934antibodies. 25934 protein can be isolated from cells or tissue sourcesusing standard protein purification techniques. 25934 protein orfragments thereof can be produced by recombinant DNA techniques orsynthesized chemically.

Polypeptides of the invention include those which arise as a result ofthe existence of multiple genes, alternative transcription events,alternative RNA splicing events, and alternative translational andpostranslational events. The polypeptide can be expressed in systems,e.g., cultured cells, which result in substantially the samepostranslational modifications present when expressed the polypeptide isexpressed in a native cell, or in systems which result in the alterationor omission of postranslational modifications, e.g., gylcosylation orcleavage, present when expressed in a native cell.

In a preferred embodiment, a 25934 polypeptide has one or more of thefollowing characteristics:

(i) it has the ability to promote the formation of a double bond atpositions up to 9 carbons from the carboxyl end of a molecule;

(ii) it has a molecular weight (e.g., a deduced molecular weight), aminoacid composition or other physical characteristic of the protein of SEQID NO:2;

(iii) it has an overall sequence similarity of at least 65-69%,preferably at least 70, 80, 90, or 95%, with a polypeptide of SEQ IDNO:2;

(iv) it has a desaturase domain which is preferably about 70%, 80%, 90%or 95% with amino acid residues 51-295 of SEQ ID NO:2;

(v) it has the fatty acid desaturase signature (ProSite PS00476) atabout residues 268 to 282 of SEQ ID NO:2;

(vi) it has eight conserved histidines which can coordinate the ironactive site at about residues 94, 99, 131, 134, 135, 272, 275, and 276of SEQ ID NO:2; or

(vii) it has at least 70%, preferably 80%, and most preferably 90% ofthe cysteines found amino acid sequence of the native protein.

In a preferred embodiment the 25934 protein, or fragment thereof,differs from the corresponding sequence in SEQ ID NO:2. In oneembodiment it differs by at least one but by less than 15, 10 or 5 aminoacid residues. In another it differs from the corresponding sequence inSEQ ID NO:2 by at least one residue but less than 20%, 15%, 10% or 5% ofthe residues in it differ from the corresponding sequence in SEQ IDNO:2. The differences are, preferably, differences or changes at anon-essential residue or a conservative substitution. In a preferredembodiment the differences are not in the desaturase domain. In anotherpreferred embodiment, one or more differences are in the desaturasedomain.

Other embodiments include a protein that contain one or more changes inamino acid sequence, e.g., a change in an amino acid residue which isnot essential for activity. Such 25934 proteins differ in amino acidsequence from SEQ ID NO:2, yet retain biological activity.

In one embodiment, the protein includes an amino acid sequence at leastabout 60%, 65%, 66-69%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or morehomologous to SEQ ID NO:2.

A 25934 protein or fragment is provided which varies from the sequenceof SEQ ID NO:2 in non-active site residues by at least one but by lessthan 15, 10 or 5 amino acid residues in the protein or fragment, butwhich does not differ from SEQ ID NO:2 in regions having desaturaseactivity. In some embodiments, the difference is at a non-essentialresidue or is a conservative substitution, while in others thedifference is at an essential residue or is a non conservativesubstitution.

In one embodiment, a biologically active portion of a 25934 proteinincludes desaturase domain. Moreover, other biologically activeportions, in which other regions of the protein are deleted, can beprepared by recombinant techniques and evaluated for one or more of thefunctional activities of a native 25934 protein.

In a preferred embodiment, the 25934 protein has an amino acid sequenceshown in SEQ ID NO:2. In other embodiments, the 25934 protein issubstantially identical to SEQ ID NO:2. In yet another embodiment, the25934 protein is substantially identical to SEQ ID NO:2 and retains thefunctional activity of the protein of SEQ ID NO:2, as described indetail in the subsections above.

25934 Chimeric or Fusion Proteins

In another aspect, the invention provides 25934 chimeric or fusionproteins. As used herein, a 25934 “chimeric protein” or “fusion protein”includes a 25934 polypeptide linked to a non-25934 polypeptide. A“non-25934 polypeptide” refers to a polypeptide having an amino acidsequence corresponding to a protein which is not substantiallyhomologous to the 25934 protein, e.g., a protein which is different fromthe 25934 protein and which is derived from the same or a differentorganism. The 25934 polypeptide of the fusion protein can correspond toall or a portion e.g., a fragment described herein of a 25934 amino acidsequence. In a preferred embodiment, a 25934 fusion protein includes atleast one (or two) biologically active portion of a 25934 protein. Thenon-25934 polypeptide can be fused to the N-terminus or C-terminus ofthe 25934 polypeptide.

The fusion protein can include a moiety which has a high affinity for aligand. For example, the fusion protein can be a GST-25934 fusionprotein in which the 25934 sequences are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification ofrecombinant 25934. Alternatively, the fusion protein can be a 25934protein containing a heterologous signal sequence at its N-terminus. Incertain host cells (e.g., mammalian host cells), expression and/orsecretion of 25934 can be increased through use of a heterologous signalsequence.

Fusion proteins can include all or a part of a serum protein, e.g., anIgG constant region, or human serum albumin.

The 25934 fusion proteins of the invention can be incorporated intopharmaceutical compositions and administered to a subject in vivo. The25934 fusion proteins can be used to affect the bioavailability of a25934 substrate. 25934 fusion proteins may be useful therapeutically forthe treatment of disorders caused by, for example, (i) aberrantmodification or mutation of a gene encoding a 25934 protein; (ii)mis-regulation of the 25934 gene; and (iii) aberrant post-translationalmodification of a 25934 protein.

Moreover, the 25934-fusion proteins of the invention can be used asimmunogens to produce anti-25934 antibodies in a subject, to purify25934 ligands and in screening assays to identify molecules whichinhibit the interaction of 25934 with a 25934 substrate.

Expression vectors are commercially available that already encode afusion moiety (e.g., a GST polypeptide). A 25934-encoding nucleic acidcan be cloned into such an expression vector such that the fusion moietyis linked in-frame to the 25934 protein.

Variants of 25934 Proteins

In another aspect, the invention also features a variant of a 25934polypeptide, e.g., which functions as an agonist (mimetics) or as anantagonist. Variants of the 25934 proteins can be generated bymutagenesis, e.g., discrete point mutation, the insertionor deletion ofsequences or the truncation of a 25934 protein. An agonist of the 25934proteins can retain substantially the same, or a subset, of thebiological activities of the naturally occurring form of a 25934protein. An antagonist of a 25934 protein can inhibit one or more of theactivities of the naturally occurring form of the 25934 protein by, forexample, competitively modulating a 25934-mediated activity of a 25934protein. Thus, specific biological effects can be elicited by treatmentwith a variant of limited function. Preferably, treatment of a subjectwith a variant having a subset of the biological activities of thenaturally occurring form of the protein has fewer side effects in asubject relative to treatment with the naturally occurring form of the25934 protein.

Variants of a 25934 protein can be identified by screening combinatoriallibraries of mutants, e.g., truncation mutants, of a 25934 protein foragonist or antagonist activity.

Libraries of fragments e.g., N terminal, C terminal, or internalfragments, of a 25934 protein coding sequence can be used to generate avariegated population of fragments for screening and subsequentselection of variants of a 25934 protein.

Variants in which a cysteine residues is added or deleted or in which aresidue which is glycosylated is added or deleted are particularlypreferred.

Methods for screening gene products of combinatorial libraries made bypoint mutations or truncation, and for screening cDNA libraries for geneproducts having a selected property. Recursive ensemble mutagenesis(REM), a new technique which enhances the frequency of functionalmutants in the libraries, can be used in combination with the screeningassays to identify 25934 variants (Arkin and Yourvan (1992) Proc. Natl.Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering6(3):327-331).

Cell based assays can be exploited to analyze a variegated 25934library. For example, a library of expression vectors can be transfectedinto a cell line, e.g., a cell line, which ordinarily responds to 25934in a substrate-dependent manner. The transfected cells are thencontacted with 25934 and the effect of the expression of the mutant onsignaling by the 25934 substrate can be detected, e.g., by measuring theformation of double bonds in the hydrocarbon areas of fatty acids.Plasmid DNA can then be recovered from the cells which score forinhibition, or alternatively, potentiation of signaling by the 25934substrate, and the individual clones further characterized.

In another aspect, the invention features a method of making a 25934polypeptide, e.g., a peptide having a non-wild type activity, e.g., anantagonist, agonist, or super agonist of a naturally occurring 25934polypeptide, e.g., a naturally occurring 25934 polypeptide. The methodincludes: altering the sequence of a 25934 polypeptide, e.g., alteringthe sequence, e.g., by substitution or deletion of one or more residuesof a non-conserved region, a domain or residue disclosed herein, andtesting the altered polypeptide for the desired activity.

In another aspect, the invention features a method of making a fragmentor analog of a 25934 polypeptide a biological activity of a naturallyoccurring 25934 polypeptide. The method includes: altering the sequence,e.g., by substitution or deletion of one or more residues, of a 25934polypeptide, e.g., altering the sequence of a non-conserved region, or adomain or residue described herein, and testing the altered polypeptidefor the desired activity.

Anti-25934 Antibodies

In another aspect, the invention provides an anti-25934 antibody. Theterm “antibody” as used herein refers to an immunoglobulin molecule orimmunologically active portion thereof, i.e., an antigen-bindingportion. Examples of immunologically active portions of immunoglobulinmolecules include F(ab) and F(ab′)₂ fragments which can be generated bytreating the antibody with an enzyme such as pepsin.

The antibody can be a polyclonal, monoclonal, recombinant, e.g., achimeric or humanized, fully human, non-human, e.g., murine, or singlechain antibody. In a preferred embodiment it has effector function andcan fix complement. The antibody can be coupled to a toxin or imagingagent.

In a preferred embodiment, the antibody fails to bind an Fc receptor,e.g., it is an isotype which does not bind to an Fc receptor, or hasbeen modified, e.g., by deletion or other mutation, such that it doesnot have a functional Fc receptor binding region.

A full-length 25934 protein or, antigenic peptide fragment of 25934 canbe used as an immunogen, or can be used to identify anti-25934antibodies made with other immunogens, e.g., cells, membranepreparations, and the like. The antigenic peptide of 25934 shouldinclude at least 8 amino acid residues of the amino acid sequence shownin SEQ ID NO:2 and encompasses an epitope of 25934. Preferably, theantigenic peptide includes at least 10 amino acid residues, morepreferably at least 15 amino acid residues, even more preferably atleast 20 amino acid residues, and most preferably at least 30 amino acidresidues.

Fragments of 25934 which include residues 30-45, 121-151, or 241-261 canbe used to make, e.g., used as immunogens, or used to characterize thespecificity of an antibody, antibodies against hydrophilic regions ofthe 25934 protein. Similarly, a fragment of 25934 which include residues291-307 can be used to make an antibody against a hydrophobic region ofthe 25934 protein; a fragment of 25934 which include all or a portion ofresidues 51-95 can be used to make an antibody against the desaturasedomain of the 25934 protein.

Antibodies reactive with, or specific for, any of these regions, orother regions or domains described herein are provided.

Preferred epitopes encompassed by the antigenic peptide are regions of25934 are located on the surface of the protein, e.g., hydrophilicregions, as well as regions with high antigenicity. For example, anEmini surface probability analysis of the human 25934 protein sequencecan be used to indicate the regions that have a particularly highprobability of being localized to the surface of the 25934 protein andare thus likely to constitute surface residues useful for targetingantibody production.

In a preferred embodiment the antibody binds an epitope on any domain orregion on 25934 proteins described herein.

Chimeric, humanized, but most preferably, completely human antibodiesare desirable for applications which include repeated administration,e.g., therapeutic treatment (and some diagnostic applications) of humanpatients.

The anti-25934 antibody can be a signle chain antibody. A single-chainantibody (scFV) may be engineered (see, for example, Colcher, D., et al.Ann N Y Acad Sci Jun. 30, 1999;880:263-80; and Reiter, Y. Clin CancerRes 1996 February;2(2):245-52). The single chain antibody can bedimerized or multimerized to generate multivalent antibodies havingspecificities for different epitopes of the same target 25934 protein.

An anti-25934 antibody (e.g., monoclonal antibody) can be used toisolate 25934 by standard techniques, such as affinity chromatography orimmunoprecipitation. Moreover, an anti-25934 antibody can be used todetect 25934 protein (e.g., in a cellular lysate or cell supernatant) inorder to evaluate the abundance and pattern of expression of theprotein. Anti-25934 antibodies can be used diagnostically to monitorprotein levels in tissue as part of a clinical testing procedure, e.g.,to, for example, determine the efficacy of a given treatment regimen.Detection can be facilitated by coupling (i.e., physically linking) theantibody to a detectable substance (i.e., antibody labelling). Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Recombinant Expression Vectors Host Cells and Genetically EngineeredCells

In another aspect, the invention includes, vectors, preferablyexpression vectors, containing a nucleic acid encoding a polypeptidedescribed herein. As used herein, the term “vector” refers to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked and can include a plasmid, cosmid or viral vector. Thevector can be capable of autonomous replication or it can integrate intoa host DNA. Viral vectors include, e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses.

A vector can include a 25934 nucleic acid in a form suitable forexpression of the nucleic acid in a host cell. Preferably therecombinant expression vector includes one or more regulatory sequencesoperatively linked to the nucleic acid sequence to be expressed. Theterm “regulatory sequence” includes promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Regulatorysequences include those which direct constitutive expression of anucleotide sequence, as well as tissue-specific regulatory and/orinducible sequences. The design of the expression vector can depend onsuch factors as the choice of the host cell to be transformed, the levelof expression of protein desired, and the like. The expression vectorsof the invention can be introduced into host cells to thereby produceproteins or polypeptides, including fusion proteins or polypeptides,encoded by nucleic acids as described herein (e.g., 25934 proteins,mutant forms of 25934 proteins, fusion proteins, and the like).

The recombinant expression vectors of the invention can be designed forexpression of 25934 proteins in prokaryotic or eukaryotic cells. Forexample, polypeptides of the invention can be expressed in E. coli,insect cells (e.g., using baculovirus expression vectors), yeast cellsor mammalian cells. Suitable host cells are discussed further inGoeddel, Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990). Alternatively, the recombinantexpression vector can be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, a proteolyticcleavage site is introduced at the junction of the fusion moiety and therecombinant protein to enable separation of the recombinant protein fromthe fusion moiety subsequent to purification of the fusion protein. Suchenzymes, and their cognate recognition sequences, include Factor Xa,thrombin and enterokinase. Typical fusion expression vectors includepGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase(GST), maltose E binding protein, or protein A, respectively, to thetarget recombinant protein.

Purified fusion proteins can be used in 25934 activity assays, (e.g.,direct assays or competitive assays described in detail below), or togenerate antibodies specific for 25934 proteins. In a preferredembodiment, a fusion protein expressed in a retroviral expression vectorof the present invention can be used to infect bone marrow cells whichare subsequently transplanted into irradiated recipients. The pathologyof the subject recipient is then examined after sufficient time haspassed (e.g., six (6) weeks).

To maximize recombinant protein expression in E. coli is to express theprotein in a host bacteria with an impaired capacity to proteolyticallycleave the recombinant protein (Gottesman, S., Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990) 119-128). Another strategy is to alter the nucleic acid sequenceof the nucleic acid to be inserted into an expression vector so that theindividual codons for each amino acid are those preferentially utilizedin E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Suchalteration of nucleic acid sequences of the invention can be carried outby standard DNA synthesis techniques.

The 25934 expression vector can be a yeast expression vector, a vectorfor expression in insect cells, e.g., a baculovirus expression vector ora vector suitable for expression in mammalian cells.

When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Non-limiting examples of suitabletissue-specific promoters include the albumin promoter (liver-specific;Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters(Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particularpromoters of T cell receptors (Winoto and Baltimore (1989) EMBO J.8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740;Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters(e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl.Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al.(1985) Science 230:912-916), and mammary gland-specific promoters (e.g.,milk whey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example, the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. Regulatory sequences (e.g., viralpromoters and/or enhancers) operatively linked to a nucleic acid clonedin the antisense orientation can be chosen which direct theconstitutive, tissue specific or cell type specific expression ofantisense RNA in a variety of cell types. The antisense expressionvector can be in the form of a recombinant plasmid, phagemid orattenuated virus. For a discussion of the regulation of gene expressionusing antisense genes see Weintraub, H. et al., Antisense RNA as amolecular tool for genetic analysis, Reviews—Trends in Genetics, Vol.1(1) 1986.

Another aspect the invention provides a host cell which includes anucleic acid molecule described herein, e.g., a 25934 nucleic acidmolecule within a recombinant expression vector or a 25934 nucleic acidmolecule containing sequences which allow it to homologously recombineinto a specific site of the host cell's genome. The terms “host cell”and “recombinant host cell” are used interchangeably herein. Such termsrefer not only to the particular subject cell but to the progeny orpotential progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

A host cell can be any prokaryotic or eukaryotic cell. For example, a25934 protein can be expressed in bacterial cells such as E. coli,insect cells, yeast or mammalian cells (such as Chinese hamster ovarycells (CHO) or COS cells). Other suitable host cells are known to thoseskilled in the art.

Vector DNA can be introduced into host cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation

A host cell of the invention can be used to produce (i.e., express) a25934 protein. Accordingly, the invention further provides methods forproducing a 25934 protein using the host cells of the invention. In oneembodiment, the method includes culturing the host cell of the invention(into which a recombinant expression vector encoding a 25934 protein hasbeen introduced) in a suitable medium such that a 25934 protein isproduced. In another embodiment, the method further includes isolating a25934 protein from the medium or the host cell.

In another aspect, the invention features, a cell or purifiedpreparation of cells which include a 25934 transgene, or which otherwisemisexpress 25934. The cell preparation can consist of human or non humancells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, orpig cells. In preferred embodiments, the cell or cells include a 25934transgene, e.g., a heterologous form of a 25934, e.g., a gene derivedfrom humans (in the case of a non-human cell). The 25934 transgene canbe misexpressed, e.g., overexpressed or underexpressed. In otherpreferred embodiments, the cell or cells include a gene which misexpressan endogenous 25934, e.g., a gene the expression of which is disrupted,e.g., a knockout. Such cells can serve as a model for studying disorderswhich are related to mutated or mis-expressed 25934 alleles or for usein drug screening.

In another aspect, the invention features, a human cell, e.g., ahematopoietic stem cell, transformed with nucleic acid which encodes asubject 25934 polypeptide.

Also provided are cells, e.g., human cells, e.g., human hematopoietic orfibroblast cells, in which an endogenous 25934 is under the control of aregulatory sequence that does not normally control the expression of theendogenous 25934 gene. The expression characteristics of an endogenousgene within a cell, e.g., a cell line or microorganism, can be modifiedby inserting a heterologous DNA regulatory element into the genome ofthe cell such that the inserted regulatory element is operably linked tothe endogenous 25934 gene. For example, an endogenous 25934 gene, e.g.,a gene that is “transcriptionally silent”, e.g., not normally expressed,or expressed only at very low levels, may be activated by inserting aregulatory element which is capable of promoting the expression of anormally expressed gene product in that cell. Techniques, such astargeted homologous recombination, can be used to insert theheterologous DNA as described in, e.g., Chappel, U.S. Pat. No.5,272,071; WO 91/06667, published in May 16, 1991.

Transgenic Animals

The invention provides non-human transgenic animals. Such animals areuseful for studying the function and/or activity of a 25934 protein andfor identifying and/or evaluating modulators of 25934 activity. As usedherein, a “transgenic animal” is a non-human animal, preferably amammal, more preferably a rodent such as a rat or mouse, in which one ormore of the cells of the animal includes a transgene. Other examples oftransgenic animals include non-human primates, sheep, dogs, cows, goats,chickens, amphibians, and the like. A transgene is exogenous DNA or arearrangment, e.g., a deletion of endogenous chromosomal DNA, whichpreferably is integrated into or occurs in the genome of the cells of atransgenic animal. A transgene can direct the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal, other transgenes, e.g., a knockout, reduce expression. Thus, atransgenic animal can be one in which an endogenous 25934 gene has beenaltered by, e.g., by homologous recombination between the endogenousgene and an exogenous DNA molecule introduced into a cell of the animal,e.g., an embryonic cell of the animal, prior to development of theanimal.

Intronic sequences and polyadenylation signals can also be included inthe transgene to increase the efficiency of expression of the transgene.A tissue-specific regulatory sequence(s) can be operably linked to atransgene of the invention to direct expression of a 25934 protein toparticular cells. A transgenic founder animal can be identified basedupon the presence of a 25934 transgene in its genome and/or expressionof 25934 mRNA in tissues or cells of the animals. A transgenic founderanimal can then be used to breed additional animals carrying thetransgene. Moreover, transgenic animals carrying a transgene encoding a25934 protein can further be bred to other transgenic animals carryingother transgenes.

25934 proteins or polypeptides can be expressed in transgenic animals orplants, e.g., a nucleic acid encoding the protein or polypeptide can beintroduced into the genome of an animal. In preferred embodiments thenucleic acid is placed under the control of a tissue specific promoter,e.g., a milk or egg specific promoter, and recovered from the milk oreggs produced by the animal. Suitable animals are mice, pigs, cows,goats, and sheep.

The invention also includes a population of cells from a transgenicanimal, as discussed, e.g., below.

Uses

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) predictive medicine (e.g., diagnostic assays,prognostic assays, monitoring clinical trials, and pharmacogenetics);and c) methods of treatment (e.g., therapeutic and prophylactic).

The isolated nucleic acid molecules of the invention can be used, forexample, to express a 25934 protein (e.g., via a recombinant expressionvector in a host cell in gene therapy applications), to detect a 25934mRNA (e.g., in a biological sample) or a genetic alteration in a 25934gene, and to modulate 25934 activity, as described further below. The25934 proteins can be used to treat disorders characterized byinsufficient or excessive production of a 25934 substrate or productionof 25934 inhibitors. In addition, the 25934 proteins can be used toscreen for naturally occurring 25934 substrates, to screen for drugs orcompounds which modulate 25934 activity, as well as to treat disorderscharacterized by insufficient or excessive production of 25934 proteinor production of 25934 protein forms which have decreased, aberrant orunwanted activity compared to 25934 wild type protein, e.g., diabetes,obesity, hypertension, cancer, developmental disorders, immune disordersand neurological and heart diseases.

Moreover, the anti-25934 antibodies of the invention can be used todetect and isolate 25934 proteins, regulate the bioavailability of 25934proteins, and modulate 25934 activity.

A method of evaluating a compound for the ability to interact with,e.g., bind, a subject 25934 polypeptide is provided. The methodincludes: contacting the compound, e.g., a substrate, with the subject25934 polypeptide; and evaluating ability of the compound to interactwith, e.g., to bind or form a complex with the subject 25934polypeptide. This method can be performed in vitro, e.g., in a cell freesystem, or in vivo, e.g., in a two-hybrid interaction trap assay. Thismethod can be used to identify naturally occurring molecules, whichinteract with subject 25934 polypeptide. It can also be used to findnatural or synthetic inhibitors of subject 25934 polypeptide. Screeningmethods are discussed in more detail below.

Screening Assays

The invention provides methods (also referred to herein as “screeningassays”) for identifying modulators, i.e., candidate or test compoundsor agents (e.g., proteins, peptides, peptidomimetics, peptoids, smallmolecules or other drugs) which bind to 25934 proteins, have astimulatory or inhibitory effect on, for example, 25934 expression or25934 activity, or have a stimulatory or inhibitory effect on, forexample, the expression or activity of a 25934 substrate. Compounds thusidentified can be used to modulate the activity of target gene products(e.g., 25934 genes) in a therapeutic protocol, to elaborate thebiological function of the target gene product, or to identify compoundsthat disrupt normal target gene interactions.

In one embodiment, the invention provides assays for screening candidateor test compounds which are substrates of a 25934 protein or polypeptideor a biologically active portion thereof. In another embodiment, theinvention provides assays for screening candidate or test compoundswhich bind to or modulate the activity of a 25934 protein or polypeptideor a biologically active portion thereof.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including: biological libraries; peptoid libraries [libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone which are resistant to enzymatic degradation butwhich nevertheless remain bioactive] (see, e.g., Zuckermann, R. N. etal. J. Med. Chem. 1994, 37: 2678-85); spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the ‘one-bead one-compound’ library method; andsynthetic library methods using affinity chromatography selection. Thebiological library and peptoid library approaches are limited to peptidelibraries, while the other four approaches are applicable to peptide,non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a 25934 protein or biologically active portion thereof iscontacted with a test compound, and the ability of the test compound tomodulate 25934 activity is determined. Determining the ability of thetest compound to modulate 25934 activity can be accomplished bymonitoring, for example, determing desaturase activity. The cell, forexample, can be of mammalian origin, e.g., a brain, ovarian, kidney,and/or liver cell.

The ability of the test compound to modulate 25934 binding to, orinteraction with, a compound, e.g., a 25934 substrate, or to bind to25934 can also be evaluated. This can be accomplished, for example, bycoupling the compound, e.g., the substrate, with a radioisotope orenzymatic label such that binding of the compound, e.g., the substrate,to 25934 can be determined by detecting the labeled compound, e.g.,substrate, in a complex. Alternatively, 25934 could be coupled with aradioisotope or enzymatic label to monitor the ability of a testcompound to modulate 25934 binding to a 25934 substrate in a complex.For example, compounds (e.g., 25934 substrates) can be labeled with¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and theradioisotope detected by direct counting of radioemmission or byscintillation counting. Alternatively, compounds can be enzymaticallylabeled with, for example, horseradish peroxidase, alkaline phosphatase,or luciferase, and the enzymatic label detected by determination ofconversion of an appropriate substrate to product.

The ability of a compound (e.g., a 25934 substrate) to interact with25934 with or without the labeling of any of the interactants can beevaluated. For example, a microphysiometer can be used to detect theinteraction of a compound with 25934 without the labeling of either thecompound or the 25934. McConnell, H. M. et al. (1992) Science257:1906-1912. As used herein, a “microphysiometer” (e.g., Cytosensor)is an analytical instrument that measures the rate at which a cellacidifies its environment using a light-addressable potentiometricsensor (LAPS). Changes in this acidification rate can be used as anindicator of the interaction between a compound and 25934.

In yet another embodiment, a cell-free assay is provided in which a25934 protein or biologically active portion thereof is contacted with atest compound and the ability of the test compound to bind to the 25934protein or biologically active portion thereof is evaluated. Preferredbiologically active portions of the 25934 proteins to be used in assaysof the present invention include fragments, which participate ininteractions with non-25934 molecules, e.g., fragments with high surfaceprobability scores.

Soluble and/or membrane-bound forms of isolated proteins (e.g., 25934proteins or biologically active portions thereof) can be used in thecell-free assays of the invention. When membrane-bound forms of theprotein are used, it may be desirable to utilize a solubilizing agent.Examples of such solubilizing agents include non-ionic detergents suchas n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylaminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

Cell-free assays involve preparing a reaction mixture of the target geneprotein and the test compound under conditions and for a time sufficientto allow the two components to interact and bind, thus forming a complexthat can be removed and/or detected.

The interaction between two molecules can also be detected, e.g., usingfluorescence energy transfer (FET) (see, for example, Lakowicz et al.,U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No.4,868,103). A fluorophore label on the first, ‘donor’ molecule isselected such that its emitted fluorescent energy will be absorbed by afluorescent label on a second, ‘acceptor’ molecule, which in turn isable to fluoresce due to the absorbed energy. Alternately, the ‘donor’protein molecule may simply utilize the natural fluorescent energy oftryptophan residues. Labels are chosen that emit different wavelengthsof light, such that the ‘acceptor’ molecule label may be differentiatedfrom that of the ‘donor’. Since the efficiency of energy transferbetween the labels is related to the distance separating the molecules,the spatial relationship between the molecules can be assessed. In asituation in which binding occurs between the molecules, the fluorescentemission of the ‘acceptor’ molecule label in the assay should bemaximal. An FET binding event can be conveniently measured throughstandard fluorometric detection means well known in the art (e.g., usinga fluorimeter).

In another embodiment, determining the ability of the 25934 protein tobind to a target molecule can be accomplished using real-timeBiomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. andUrbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995)Curr. Opin. Struct. Biol. 5:699-705). “Surface plasmon resonance” or“BIA” detects biospecific interactions in real time, without labelingany of the interactants (e.g., BIAcore). Changes in the mass at thebinding surface (indicative of a binding event) result in alterations ofthe refractive index of light near the surface (the optical phenomenonof surface plasmon resonance (SPR)), resulting in a detectable signalwhich can be used as an indication of real-time reactions betweenbiological molecules.

In one embodiment, the target gene product or the test substance isanchored onto a solid phase. The target gene product/test compoundcomplexes anchored on the solid phase can be detected at the end of thereaction. Preferably, the target gene product can be anchored onto asolid surface, and the test compound, (which is not anchored), can belabeled, either directly or indirectly, with detectable labels discussedherein.

It may be desirable to immobilize either 25934, an anti-25934 antibodyor its target molecule to facilitate separation of complexed fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a test compound to a25934 protein, or interaction of a 25934 protein with a target moleculein the presence and absence of a candidate compound, can be accomplishedin any vessel suitable for containing the reactants. Examples of suchvessels include microtiter plates, test tubes, and micro-centrifugetubes. In one embodiment, a fusion protein can be provided which adds adomain that allows one or both of the proteins to be bound to a matrix.For example, glutathione-S-transferase/25934 fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or 25934 protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level of 25934binding or activity determined using standard techniques.

Other techniques for immobilizing either a 25934 protein or a targetmolecule on matrices include using conjugation of biotin andstreptavidin. Biotinylated 25934 protein or target molecules can beprepared from biotin-NHS (N-hydroxy-succinimide) using techniques knownin the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.),and immobilized in the wells of streptavidin-coated 96 well plates(Pierce Chemical).

In order to conduct the assay, the non-immobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynon-immobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously non-immobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the immobilized component (theantibody, in turn, can be directly labeled or indirectly labeled with,e.g., a labeled anti-Ig antibody).

In one embodiment, this assay is performed utilizing antibodies reactivewith 25934 protein or target molecules but which do not interfere withbinding of the 25934 protein to its target molecule. Such antibodies canbe derivatized to the wells of the plate, and unbound target or 25934protein trapped in the wells by antibody conjugation. Methods fordetecting such complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the 25934 protein or target molecule, as wellas enzyme-linked assays which rely on detecting an enzymatic activityassociated with the 25934 protein or target molecule.

Alternatively, cell free assays can be conducted in a liquid phase. Insuch an assay, the reaction products are separated from unreactedcomponents, by any of a number of standard techniques, including but notlimited to: differential centrifugation (see, for example, Rivas, G.,and Minton, A. P., Trends Biochem Sci 1993 August;18(8):284-7);chromatography (gel filtration chromatography, ion-exchangechromatography); electrophoresis (see, e.g., Ausubel, F. et al., eds.Current Protocols in Molecular Biology 1999, J. Wiley: New York.); andimmunoprecipitation (see, for example, Ausubel, F. et al., eds. CurrentProtocols in Molecular Biology 1999, J. Wiley: New York). Such resinsand chromatographic techniques are known to one skilled in the art (see,e.g., Heegaard, N. H., J Mol Recognit 1998 Winter;11(1-6):141-8; Hage,D. S., and Tweed, S. A. J Chromatogr B Biomed Sci Appl Oct. 10,1997;699(1-2):499-525). Further, fluorescence energy transfer may alsobe conveniently utilized, as described herein, to detect binding withoutfurther purification of the complex from solution.

In a preferred embodiment, the assay includes contacting the 25934protein or biologically active portion thereof with a known compoundwhich binds 25934 to form an assay mixture, contacting the assay mixturewith a test compound, and determining the ability of the test compoundto interact with a 25934 protein, wherein determining the ability of thetest compound to interact with a 25934 protein includes determining theability of the test compound to preferentially bind to 25934 orbiologically active portion thereof, or to modulate the activity of atarget molecule, as compared to the known compound.

The target gene products of the invention can, in vivo, interact withone or more cellular or extracellular macromolecules, such as proteins.For the purposes of this discussion, such cellular and extracellularmacromolecules are referred to herein as “binding partners.” Compoundsthat disrupt such interactions can be useful in regulating the activityof the target gene product. Such compounds can include, but are notlimited to molecules such as antibodies, peptides, and small molecules.The preferred target genes/products for use in this embodiment are the25934 genes herein identified. In an alternative embodiment, theinvention provides methods for determining the ability of the testcompound to modulate the activity of a 25934 protein through modulationof the activity of a downstream effector of a 25934 target molecule. Forexample, the activity of the effector molecule on an appropriate targetcan be determined, or the binding of the effector to an appropriatetarget can be determined, as previously described.

To identify compounds that interfere with the interaction between thetarget gene product and its cellular or extracellular bindingpartner(s), a reaction mixture containing the target gene product andthe binding partner is prepared, under conditions and for a timesufficient, to allow the two products to form complex. In order to testan inhibitory agent, the reaction mixture is provided in the presenceand absence of the test compound. The test compound can be initiallyincluded in the reaction mixture, or can be added at a time subsequentto the addition of the target gene and its cellular or extracellularbinding partner. Control reaction mixtures are incubated without thetest compound or with a placebo. The formation of any complexes betweenthe target gene product and the cellular or extracellular bindingpartner is then detected. The formation of a complex in the controlreaction, but not in the reaction mixture containing the test compound,indicates that the compound interferes with the interaction of thetarget gene product and the interactive binding partner. Additionally,complex formation within reaction mixtures containing the test compoundand normal target gene product can also be compared to complex formationwithin reaction mixtures containing the test compound and mutant targetgene product. This comparison can be important in those cases wherein itis desirable to identify compounds that disrupt interactions of mutantbut not normal target gene products.

These assays can be conducted in a heterogeneous or homogeneous format.Heterogeneous assays involve anchoring either the target gene product orthe binding partner onto a solid phase, and detecting complexes anchoredon the solid phase at the end of the reaction. In homogeneous assays,the entire reaction is carried out in a liquid phase. In eitherapproach, the order of addition of reactants can be varied to obtaindifferent information about the compounds being tested. For example,test compounds that interfere with the interaction between the targetgene products and the binding partners, e.g., by competition, can beidentified by conducting the reaction in the presence of the testsubstance. Alternatively, test compounds that disrupt preformedcomplexes, e.g., compounds with higher binding constants that displaceone of the components from the complex, can be tested by adding the testcompound to the reaction mixture after complexes have been formed. Thevarious formats are briefly described below.

In a heterogeneous assay system, either the target gene product or theinteractive cellular or extracellular binding partner, is anchored ontoa solid surface (e.g., a microtiter plate), while the non-anchoredspecies is labeled, either directly or indirectly. The anchored speciescan be immobilized by non-covalent or covalent attachments.Alternatively, an immobilized antibody specific for the species to beanchored can be used to anchor the species to the solid surface.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. Where the non-immobilized species is pre-labeled, the detectionof label immobilized on the surface indicates that complexes wereformed. Where the non-immobilized species is not pre-labeled, anindirect label can be used to detect complexes anchored on the surface;e.g., using a labeled antibody specific for the initiallynon-immobilized species (the antibody, in turn, can be directly labeledor indirectly labeled with, e.g., a labeled anti-Ig antibody). Dependingupon the order of addition of reaction components, test compounds thatinhibit complex formation or that disrupt preformed complexes can bedetected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds that inhibit complex or that disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. For example, a preformed complex of the target gene product andthe interactive cellular or extracellular binding partner product isprepared in that either the target gene products or their bindingpartners are labeled, but the signal generated by the label is quencheddue to complex formation (see, e.g., U.S. Pat. No. 4,109,496 thatutilizes this approach for immunoassays). The addition of a testsubstance that competes with and displaces one of the species from thepreformed complex will result in the generation of a signal abovebackground. In this way, test substances that disrupt target geneproduct-binding partner interaction can be identified.

In yet another aspect, the 25934 proteins can be used as “bait proteins”in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No.5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J.Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and BrentWO94/10300), to identify other proteins, which bind to or interact with25934 (“25934-binding proteins” or “25934-bp”) and are involved in 25934activity. Such 25934-bps can be activators or inhibitors of signals bythe 25934 proteins or 25934 targets as, for example, downstream elementsof a 25934-mediated signaling pathway.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a 25934 protein isfused to a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences, that encodes an unidentified protein (“prey”or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. (Alternatively the: 25934 protein can bethe fused to the activator domain.) If the “bait” and the “prey”proteins are able to interact, in vivo, forming a 25934-dependentcomplex, the DNA-binding and activation domains of the transcriptionfactor are brought into close proximity. This proximity allowstranscription of a reporter gene (e.g., LacZ) which is operably linkedto a transcriptional regulatory site responsive to the transcriptionfactor. Expression of the reporter gene can be detected and cellcolonies containing the functional transcription factor can be isolatedand used to obtain the cloned gene which encodes the protein whichinteracts with the 25934 protein.

In another embodiment, modulators of 25934 expression are identified.For example, a cell or cell free mixture is contacted with a candidatecompound and the expression of 25934 mRNA or protein evaluated relativeto the level of expression of 25934 mRNA or protein in the absence ofthe candidate compound. When expression of 25934 mRNA or protein isgreater in the presence of the candidate compound than in its absence,the candidate compound is identified as a stimulator of 25934 mRNA orprotein expression. Alternatively, when expression of 25934 mRNA orprotein is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound isidentified as an inhibitor of 25934 mRNA or protein expression. Thelevel of 25934 mRNA or protein expression can be determined by methodsdescribed herein for detecting 25934 mRNA or protein.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell free assay, and the abilityof the agent to modulate the activity of a 25934 protein can beconfirmed in vivo, e.g., using an animal model.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein(e.g., a 25934 modulating agent, an antisense 25934 nucleic acidmolecule, a 25934-specific antibody, or a 25934-binding partner) in anappropriate animal model to determine the efficacy, toxicity, sideeffects, or mechanism of action, of treatment with such an agent.Furthermore, novel agents identified by the above-described screeningassays can be used for treatments as described herein.

Detection Assays

Portions or fragments of the nucleic acid sequences identified hereincan be used as polynucleotide reagents. For example, these sequences canbe used to: (i) map their respective genes on a chromosome e.g., tolocate gene regions associated with genetic disease or to associate25934 with a disease; (ii) identify an individual from a minutebiological sample (tissue typing); and (iii) aid in forensicidentification of a biological sample. These applications are describedin the subsections below.

Chromosome Mapping

The 25934 nucleotide sequences or portions thereof can be used to mapthe location of the 25934 genes on a chromosome. This process is calledchromosome mapping. Chromosome mapping is useful in correlating the25934 sequences with genes associated with disease.

Briefly, 25934 genes can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp in length) from the 25934 nucleotidesequences. These primers can then be used for PCR screening of somaticcell hybrids containing individual human chromosomes. Only those hybridscontaining the human gene corresponding to the 25934 sequences willyield an amplified fragment.

A panel of somatic cell hybrids in which each cell line contains eithera single human chromosome or a small number of human chromosomes, and afull set of mouse chromosomes, can allow easy mapping of individualgenes to specific human chromosomes. (D'Eustachio P. et al. (1983)Science 220:919-924).

Other mapping strategies e.g., in situ hybridization (described in Fan,Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screeningwith labeled flow-sorted chromosomes, and pre-selection by hybridizationto chromosome specific cDNA libraries can be used to map 25934 to achromosomal location.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. The FISH technique can be used with aDNA sequence as short as 500 or 600 bases. However, clones larger than1,000 bases have a higher likelihood of binding to a unique chromosomallocation with sufficient signal intensity for simple detection.Preferably 1,000 bases, and more preferably 2,000 bases will suffice toget good results at a reasonable amount of time. For a review of thistechnique, see Verma et al., Human Chromosomes: A Manual of BasicTechniques (Pergamon Press, New York 1988).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship between agene and a disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, for example, Egeland, J. et al. (1987)Nature, 325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the 25934 gene, can bedetermined. If a mutation is observed in some or all of the affectedindividuals but not in any unaffected individuals, then the mutation islikely to be the causative agent of the particular disease. Comparisonof affected and unaffected individuals generally involves first lookingfor structural alterations in the chromosomes, such as deletions ortranslocations that are visible from chromosome spreads or detectableusing PCR based on that DNA sequence. Ultimately, complete sequencing ofgenes from several individuals can be performed to confirm the presenceof a mutation and to distinguish mutations from polymorphisms.

Tissue Typing

25934 sequences can be used to identify individuals from biologicalsamples using, e.g., restriction fragment length polymorphism (RFLP). Inthis technique, an individual's genomic DNA is digested with one or morerestriction enzymes, the fragments separated, e.g., in a Southern blot,and probed to yield bands for identification. The sequences of thepresent invention are useful as additional DNA markers for RFLP(described in U.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can also be used todetermine the actual base-by-base DNA sequence of selected portions ofan individual's genome. Thus, the 25934 nucleotide sequences describedherein can be used to prepare two PCR primers from the 5′ and 3′ ends ofthe sequences. These primers can then be used to amplify an individual'sDNA and subsequently sequence it. Panels of corresponding DNA sequencesfrom individuals, prepared in this manner, can provide unique individualidentifications, as each individual will have a unique set of such DNAsequences due to allelic differences.

Allelic variation occurs to some degree in the coding regions of thesesequences, and to a greater degree in the noncoding regions. Each of thesequences described herein can, to some degree, be used as a standardagainst which DNA from an individual can be compared for identificationpurposes. Because greater numbers of polymorphisms occur in thenoncoding regions, fewer sequences are necessary to differentiateindividuals. The noncoding sequences of SEQ ID NO:1 can provide positiveindividual identification with a panel of perhaps 10 to 1,000 primerswhich each yield a noncoding amplified sequence of 100 bases. Ifpredicted coding sequences, such as those in SEQ ID NO:3 are used, amore appropriate number of primers for positive individualidentification would be 500-2,000.

If a panel of reagents from 25934 nucleotide sequences described hereinis used to generate a unique identification database for an individual,those same reagents can later be used to identify tissue from thatindividual. Using the unique identification database, positiveidentification of the individual, living or dead, can be made fromextremely small tissue samples.

Use of Partial 25934 Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. To make such an identification, PCR technology can be used toamplify DNA sequences taken from very small biological samples such astissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, orsemen found at a crime scene. The amplified sequence can then becompared to a standard, thereby allowing identification of the origin ofthe biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NO:1 (e.g., fragments derivedfrom the noncoding regions of SEQ ID NO:1 having a length of at least 20bases, preferably at least 30 bases) are particularly appropriate forthis use.

The 25934 nucleotide sequences described herein can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes whichcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue, e.g., a tissue containing 25934. This can bevery useful in cases where a forensic pathologist is presented with atissue of unknown origin. Panels of such 25934 probes can be used toidentify tissue by species and/or by organ type.

In a similar fashion, these reagents, e.g., 25934 primers or probes canbe used to screen tissue culture for contamination (i.e. screen for thepresence of a mixture of different types of cells in a culture).

Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring clinicaltrials are used for prognostic (predictive) purposes to thereby treat anindividual.

Generally, the invention provides, a method of determining if a subjectis at risk for a disorder related to a lesion in or the misexpression ofa gene which encodes 25934.

Such disorders include, e.g., a disorder associated with themisexpression 25934; a disorder associated with unsaturation of fattyacids, e.g., cardiovascular, diabetes, obesity, hypertension, cancer,and neurological diseases.

The method includes one or more of the following:

detecting, in a tissue of the subject, the presence or absence of amutation which affects the expression of the 25934 gene, or detectingthe presence or absence of a mutation in a region which controls theexpression of the gene, e.g., a mutation in the 5′ control region;

detecting, in a tissue of the subject, the presence or absence of amutation which alters the structure of the 25934 gene;

detecting, in a tissue of the subject, the misexpression of the 25934gene, at the mRNA level, e.g., detecting a non-wild type level of amRNA;

detecting, in a tissue of the subject, the misexpression of the gene, atthe protein level, e.g., detecting a non-wild type level of a 25934polypeptide.

In preferred embodiments the method includes: ascertaining the existenceof at least one of: a deletion of one or more nucleotides from the 25934gene; an insertion of one or more nucleotides into the gene, a pointmutation, e.g., a substitution of one or more nucleotides of the gene, agross chromosomal rearrangement of the gene, e.g., a translocation,inversion, or deletion.

For example, detecting the genetic lesion can include: (i) providing aprobe/primer including an oligonucleotide containing a region ofnucleotide sequence which hybridizes to a sense or antisense sequencefrom SEQ ID NO:1, or naturally occurring mutants thereof or 5′ or 3′flanking sequences naturally associated with the 25934 gene; (ii)exposing the probe/primer to nucleic acid of the tissue; and detecting,by hybridization, e.g., in situ hybridization, of the probe/primer tothe nucleic acid, the presence or absence of the genetic lesion.

In preferred embodiments detecting the misexpression includesascertaining the existence of at least one of: an alteration in thelevel of a messenger RNA transcript of the 2593 gene; the presence of anon-wild type splicing pattern of a messenger RNA transcript of thegene; or a non-wild type level of 25934.

Methods of the invention can be used prenatally or to determine if asubject's offspring will be at risk for a disorder.

In preferred embodiments the method includes determining the structureof a 25934 gene, an abnormal structure being indicative of risk for thedisorder.

In preferred embodiments the method includes contacting a sample formthe subject with an antibody to the 25934 protein or a nucleic acid,which hybridizes specifically with the gene. There and other embodimentsare discussed below.

Diagnostic and Prognostic Assays

The presence, level, or absence of 25934 protein or nucleic acid in abiological sample can be evaluated by obtaining a biological sample froma test subject and contacting the biological sample with a compound oran agent capable of detecting 25934 protein or nucleic acid (e.g., mRNA,genomic DNA) that encodes 25934 protein such that the presence of 25934protein or nucleic acid is detected in the biological sample. The term“biological sample” includes tissues, cells and biological fluidsisolated from a subject, as well as tissues, cells and fluids presentwithin a subject. A preferred biological sample is serum. The level ofexpression of the 25934 gene can be measured in a number of ways,including, but not limited to: measuring the mRNA encoded by the 25934genes; measuring the amount of protein encoded by the 25934 genes; ormeasuring the activity of the protein encoded by the 25934 genes.

The level of mRNA corresponding to the 25934 gene in a cell can bedetermined both by in situ and by in vitro formats.

The isolated mRNA can be used in hybridization or amplification assaysthat include, but are not limited to, Southern or Northern analyses,polymerase chain reaction analyses and probe arrays. One preferreddiagnostic method for the detection of mRNA levels involves contactingthe isolated mRNA with a nucleic acid molecule (probe) that canhybridize to the mRNA encoded by the gene being detected. The nucleicacid probe can be, for example, a full-length 25934 nucleic acid, suchas the nucleic acid of SEQ ID NO:1, or the DNA insert of the plasmiddeposited with ATCC as Accession Number 2167, or a portion thereof, suchas an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500nucleotides in length and sufficient to specifically hybridize understringent conditions to 25934 mRNA or genomic DNA. The probe can bedisposed on an address of an array, e.g., an array described below.Other suitable probes for use in the diagnostic assays are describedherein.

In one format, mRNA (or cDNA) is immobilized on a surface and contactedwith the probes, for example by running the isolated mRNA on an agarosegel and transferring the mRNA from the gel to a membrane, such asnitrocellulose. In an alternative format, the probes are immobilized ona surface and the mRNA (or cDNA) is contacted with the probes, forexample, in a two-dimensional gene chip array. A skilled artisan canadapt known mRNA detection methods for use in detecting the level ofmRNA encoded by the 25934 genes.

The level of mRNA in a sample that is encoded by one of 25934 can beevaluated with nucleic acid amplification, e.g., by rtPCR (Mullis, 1987,U.S. Pat. No. 4,683,202), ligase chain reaction (Barany, 1991, Proc.Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication(Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878),transcriptional amplification system (Kwoh et al., 1989, Proc. Natl.Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988,Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S.Pat. No. 5,854,033) or any other nucleic acid amplification method,followed by the detection of the amplified molecules using techniquesknown in the art. As used herein, amplification primers are defined asbeing a pair of nucleic acid molecules that can anneal to 5′ or 3′regions of a gene (plus and minus strands, respectively, or vice-versa)and contain a short region in between. In general, amplification primersare from about 10 to 30 nucleotides in length and flank a region fromabout 50 to 200 nucleotides in length. Under appropriate conditions andwith appropriate reagents, such primers permit the amplification of anucleic acid molecule comprising the nucleotide sequence flanked by theprimers.

For in situ methods, a cell or tissue sample can be prepared/processedand immobilized on a support, typically a glass slide, and thencontacted with a probe that can hybridize to mRNA that encodes the 25934gene being analyzed.

In another embodiment, the methods further contacting a control samplewith a compound or agent capable of detecting 25934 mRNA, or genomicDNA, and comparing the presence of 25934 mRNA or genomic DNA in thecontrol sample with the presence of 25934 mRNA or genomic DNA in thetest sample.

A variety of methods can be used to determine the level of proteinencoded by 25934. In general, these methods include contacting an agentthat selectively binds to the protein, such as an antibody with asample, to evaluate the level of protein in the sample. In a preferredembodiment, the antibody bears a detectable label. Antibodies can bepolyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof (e.g., Fab or F(ab′)₂) can be used. The term “labeled”,with regard to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by coupling (i.e., physically linking)a detectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with a detectablesubstance. Examples of detectable substances are provided herein.

The detection methods can be used to detect 25934 protein in abiological sample in vitro as well as in vivo. In vitro techniques fordetection of 25934 protein include enzyme linked immunosorbent assays(ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay(EIA), radioimmunoassay (RIA), and Western blot analysis. In vivotechniques for detection of 25934 protein include introducing into asubject a labeled anti-25934 antibody. For example, the antibody can belabeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques.

In another embodiment, the methods further include contacting thecontrol sample with a compound or agent capable of detecting 25934protein, and comparing the presence of 25934 protein in the controlsample with the presence of 25934 protein in the test sample.

The invention also includes kits for detecting the presence of 25934 ina biological sample. For example, the kit can include a compound oragent capable of detecting 25934 protein or mRNA in a biological sample;and a standard. The compound or agent can be packaged in a suitablecontainer. The kit can further comprise instructions for using the kitto detect 25934 protein or nucleic acid.

For antibody-based kits, the kit can include: (1) a first antibody(e.g., attached to a solid support) which binds to a polypeptidecorresponding to a marker of the invention; and, optionally, (2) asecond, different antibody which binds to either the polypeptide or thefirst antibody and is conjugated to a detectable agent.

For oligonucleotide-based kits, the kit can include: (1) anoligonucleotide, e.g., a detectably labeled oligonucleotide, whichhybridizes to a nucleic acid sequence encoding a polypeptidecorresponding to a marker of the invention or (2) a pair of primersuseful for amplifying a nucleic acid molecule corresponding to a markerof the invention. The kit can also includes a buffering agent, apreservative, or a protein stabilizing agent. The kit can also includescomponents necessary for detecting the detectable agent (e.g., an enzymeor a substrate). The kit can also contain a control sample or a seriesof control samples which can be assayed and compared to the test samplecontained. Each component of the kit can be enclosed within anindividual container and all of the various containers can be within asingle package, along with instructions for interpreting the results ofthe assays performed using the kit.

The diagnostic methods described herein can identify subjects having, orat risk of developing, a disease or disorder associated withmisexpressed or aberrant or unwanted 25934 expression or activity. Asused herein, the term “unwanted” includes an unwanted phenomenoninvolved in a biological response such as pain or deregulated cellproliferation.

In one embodiment, a disease or disorder associated with aberrant orunwanted 25934 expression or activity is identified. A test sample isobtained from a subject and 25934 protein or nucleic acid (e.g., mRNA orgenomic DNA) is evaluated, wherein the level, e.g., the presence orabsence, of 25934 protein or nucleic acid is diagnostic for a subjecthaving or at risk of developing a disease or disorder associated withaberrant or unwanted 25934 expression or activity. As used herein, a“test sample” refers to a biological sample obtained from a subject ofinterest, including a biological fluid (e.g., serum), cell sample, ortissue.

The prognostic assays described herein can be used to determine whethera subject can be administered an agent (e.g., an agonist, antagonist,peptidomimetic, protein, peptide, nucleic acid, small molecule, or otherdrug candidate) to treat a disease or disorder associated with aberrantor unwanted 25934 expression or activity. For example, such methods canbe used to determine whether a subject can be effectively treated withan agent for a cell disorder associated with abnormal or abherrantunsaturation of fatty acids.

The methods of the invention can also be used to detect geneticalterations in a 25934 gene, thereby determining if a subject with thealtered gene is at risk for a disorder characterized by misregulation in25934 protein activity or nucleic acid expression, such as as in thefollowing disorders: cardiovascular, diabetes, obesity, hypertension,cancer, developmental disorders, immune disorders and neurologicaldiseases.

In preferred embodiments, the methods include detecting, in a samplefrom the subject, the presence or absence of a genetic alterationcharacterized by at least one of an alteration affecting the integrityof a gene encoding a 25934-protein, or the mis-expression of the 25934gene. For example, such genetic alterations can be detected byascertaining the existence of at least one of 1) a deletion of one ormore nucleotides from a 25934 gene; 2) an addition of one or morenucleotides to a 25934 gene; 3) a substitution of one or morenucleotides of a 25934 gene, 4) a chromosomal rearrangement of a 25934gene; 5) an alteration in the level of a messenger RNA transcript of a25934 gene, 6) aberrant modification of a 25934 gene, such as of themethylation pattern of the genomic DNA, 7) the presence of a non-wildtype splicing pattern of a messenger RNA transcript of a 25934 gene, 8)a non-wild type level of a 25934-protein, 9) allelic loss of a 25934gene, and 10) inappropriate post-translational modification of a25934-protein.

An alteration can be detected without a probe/primer in a polymerasechain reaction, such as anchor PCR or RACE PCR, or, alternatively, in aligation chain reaction (LCR), the latter of which can be particularlyuseful for detecting point mutations in the 25934-gene. This method caninclude the steps of collecting a sample of cells from a subject,isolating nucleic acid (e.g., genomic, mRNA or both) from the sample,contacting the nucleic acid sample with one or more primers whichspecifically hybridize to a 25934 gene under conditions such thathybridization and amplification of the 25934-gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.,(1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al. (1988) Bio-Technology 6:1197), or other nucleicacid amplification methods, followed by the detection of the amplifiedmolecules using techniques known to those of skill in the art.

In another embodiment, mutations in a 25934 gene from a sample cell canbe identified by detecting alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined, e.g., by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in 25934 can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, twodimensional arrays, e.g., chip based arrays. Such arrays include aplurality of addresses, each of which is positionally distinguishablefrom the other. A different probe is located at each address of theplurality. The arrays can have a high density of addresses, e.g., cancontain hundreds or thousands of oligonucleotides probes (Cronin, M. T.et al. (1996) Human Mutation 7: 244-255; Kozal, M. J. et al. (1996)Nature Medicine 2: 753-759). For example, genetic mutations in 25934 canbe identified in two dimensional arrays containing light-generated DNAprobes as described in Cronin, M. T. et al. supra. Briefly, a firsthybridization array of probes can be used to scan through long stretchesof DNA in a sample and control to identify base changes between thesequences by making linear arrays of sequential overlapping probes. Thisstep allows the identification of point mutations. This step is followedby a second hybridization array that allows the characterization ofspecific mutations by using smaller, specialized probe arrayscomplementary to all variants or mutations detected. Each mutation arrayis composed of parallel probe sets, one complementary to the wild-typegene and the other complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the 25934 gene anddetect mutations by comparing the sequence of the sample 25934 with thecorresponding wild-type (control) sequence. Automated sequencingprocedures can be utilized when performing the diagnostic assays ((1995)Biotechniques 19:448), including sequencing by mass spectrometry.

Other methods for detecting mutations in the 25934 gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242; Cotton et al. (1988) Proc. Natl Acad Sci USA 85:4397; Saleebaet al. (1992) Methods Enzymol. 217:286-295).

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in 25934 cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662; U.S.Pat. No. 5,459,039).

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in 25934 genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton(1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech.Appl. 9:73-79). Single-stranded DNA fragments of sample and control25934 nucleic acids will be denatured and allowed to renature. Thesecondary structure of single-stranded nucleic acids varies according tosequence, the resulting alteration in electrophoretic mobility enablesthe detection of even a single base change. The DNA fragments may belabeled or detected with labeled probes. The sensitivity of the assaymay be enhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys Chem 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension (Saiki et al. (1986) Nature324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230).

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a 25934 gene.

Use of 25934 Molecules as Surrogate Markers

The 25934 molecules of the invention are also useful as markers ofdisorders or disease states, as markers for precursors of diseasestates, as markers for predisposition of disease states, as markers ofdrug activity, or as markers of the pharmacogenomic profile of asubject. Using the methods described herein, the presence, absenceand/or quantity of the 25934 molecules of the invention may be detected,and may be correlated with one or more biological states in vivo. Forexample, the 25934 molecules of the invention may serve as surrogatemarkers for one or more disorders or disease states or for conditionsleading up to disease states. As used herein, a “surrogate marker” is anobjective biochemical marker which correlates with the absence orpresence of a disease or disorder, or with the progression of a diseaseor disorder (e.g., with the presence or absence of a tumor). Thepresence or quantity of such markers is independent of the disease.Therefore, these markers may serve to indicate whether a particularcourse of treatment is effective in lessening a disease state ordisorder. Surrogate markers are of particular use when the presence orextent of a disease state or disorder is difficult to assess throughstandard methodologies (e.g., early stage tumors), or when an assessmentof disease progression is desired before a potentially dangerousclinical endpoint is reached (e.g., an assessment of cardiovasculardisease may be made using cholesterol levels as a surrogate marker, andan analysis of HIV infection may be made using HIV RNA levels as asurrogate marker, well in advance of the undesirable clinical outcomesof myocardial infarction or fully-developed AIDS). Examples of the useof surrogate markers in the art include: Koomen et al. (2000) J. Mass.Spectrom. 35: 258-264; and James (1994) AIDS Treatment News Archive 209.

The 25934 molecules of the invention are also useful as pharmacodynamicmarkers. As used herein, a “pharmacodynamic marker” is an objectivebiochemical marker which correlates specifically with drug effects. Thepresence or quantity of a pharmacodynamic marker is not related to thedisease state or disorder for which the drug is being administered;therefore, the presence or quantity of the marker is indicative of thepresence or activity of the drug in a subject. For example, apharmacodynamic marker may be indicative of the concentration of thedrug in a biological tissue, in that the marker is either expressed ortranscribed or not expressed or transcribed in that tissue inrelationship to the level of the drug. In this fashion, the distributionor uptake of the drug may be monitored by the pharmacodynamic marker.Similarly, the presence or quantity of the pharmacodynamic marker may berelated to the presence or quantity of the metabolic product of a drug,such that the presence or quantity of the marker is indicative of therelative breakdown rate of the drug in vivo. Pharmacodynamic markers areof particular use in increasing the sensitivity of detection of drugeffects, particularly when the drug is administered in low doses. Sinceeven a small amount of a drug may be sufficient to activate multiplerounds of marker (e.g., a 25934 marker) transcription or expression, theamplified marker may be in a quantity which is more readily detectablethan the drug itself. Also, the marker may be more easily detected dueto the nature of the marker itself; for example, using the methodsdescribed herein, anti-25934 antibodies may be employed in animmune-based detection system for a 25934 protein marker, or25934-specific radiolabeled probes may be used to detect a 25934 mRNAmarker. Furthermore, the use of a pharmacodynamic marker may offermechanism-based prediction of risk due to drug treatment beyond therange of possible direct observations. Examples of the use ofpharmacodynamic markers in the art include: Matsuda et al. U.S. Pat. No.6,033,862; Hattis et al. (1991) Env. Health Perspect. 90: 229-238;Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3: S21-S24; andNicolau (1999) Am, J. Health-Syst. Pharm. 56 Suppl. 3:S16-S20.

The 25934 molecules of the invention are also useful as pharmacogenomicmarkers. As used herein, a “pharmacogenomic marker” is an objectivebiochemical marker which correlates with a specific clinical drugresponse or susceptibility in a subject (see, e.g., McLeod et al. (1999)Eur. J. Cancer 35(12): 1650-1652). The presence or quantity of thepharmacogenomic marker is related to the predicted response of thesubject to a specific drug or class of drugs prior to administration ofthe drug. By assessing the presence or quantity of one or morepharmacogenomic markers in a subject, a drug therapy which is mostappropriate for the subject, or which is predicted to have a greaterdegree of success, may be selected. For example, based on the presenceor quantity of RNA, or protein (e.g., 25934 protein or RNA) for specifictumor markers in a subject, a drug or course of treatment may beselected that is optimized for the treatment of the specific tumorlikely to be present in the subject. Similarly, the presence or absenceof a specific sequence mutation in 25934 DNA may correlate 25934 drugresponse. The use of pharmacogenomic markers therefore permits theapplication of the most appropriate treatment for each subject withouthaving to administer the therapy.

Pharmaceutical Compositions

The nucleic acid and polypeptides, fragments thereof, as well asanti-25934 antibodies (also referred to herein as “active compounds”) ofthe invention can be incorporated into pharmaceutical compositions. Suchcompositions typically include the nucleic acid molecule, protein, orantibody and a pharmaceutically acceptable carrier. As used herein thelanguage “pharmaceutically acceptable carrier” includes solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Supplementary active compounds can alsobe incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oral(e.g., inhalation), transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit high therapeutic indeces are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The protein or polypeptide can be administered onetime per week for between about 1 to 10 weeks, preferably between 2 to 8weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. The skilled artisan willappreciate that certain factors may influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, previous treatments, thegeneral health and/or age of the subject, and other diseases present.Moreover, treatment of a subject with a therapeutically effective amountof a protein, polypeptide, or antibody can include a single treatmentor, preferably, can include a series of treatments.

For antibodies, the preferred dosage is 0.1 mg/kg of body weight(generally 10 mg/kg to 20 mg/kg). If the antibody is to act in thebrain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into thebrain). A method for lipidation of antibodies is described by Cruikshanket al. ((1997) J. Acquired Immune Deficiency Syndromes and HumanRetrovirology 14:193).

The present invention encompasses agents which modulate expression oractivity. An agent may, for example, be a small molecule. For example,such small molecules include, but are not limited to, peptides,peptidomimetics (e.g., peptoids), amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic or inorganic compounds (i.e., including heteroorganicand organometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds.

Exemplary doses include milligram or microgram amounts of the smallmolecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram. It isfurthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. When one or more of these small molecules isto be administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

An antibody (or fragment thereof) may be conjugated to a therapeuticmoiety such as a cytotoxin, a therapeutic agent or a radioactive metalion. A cytotoxin or cytotoxic agent includes any agent that isdetrimental to cells. Examples include taxol, cytochalasin B, gramicidinD, ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, .alpha.-interferon, .beta.-interferon, nervegrowth factor, platelet derived growth factor, tissue plasminogenactivator; or, biological response modifiers such as, for example,lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant or unwanted 25934expression or activity. With regards to both prophylactic andtherapeutic methods of treatment, such treatments may be specificallytailored or modified, based on knowledge obtained from the field ofpharmacogenomics. “Pharmacogenomics”, as used herein, refers to theapplication of genomics technologies such as gene sequencing,statistical genetics, and gene expression analysis to drugs in clinicaldevelopment and on the market. More specifically, the term refers thestudy of how a patient's genes determine his or her response to a drug(e.g., a patient's “drug response phenotype”, or “drug responsegenotype”.) Thus, another aspect of the invention provides methods fortailoring an individual's prophylactic or therapeutic treatment witheither the 25934 molecules of the present invention or 25934 modulatorsaccording to that individual's drug response genotype. Pharmacogenomicsallows a clinician or physician to target prophylactic or therapeutictreatments to patients who will most benefit from the treatment and toavoid treatment of patients who will experience toxic drug-related sideeffects.

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant or unwanted25934 expression or activity, by administering to the subject a 25934 oran agent which modulates 25934 expression or at least one 25934activity. Subjects at risk for a disease which is caused or contributedto by aberrant or unwanted 25934 expression or activity can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe 25934 aberrance, such that a disease or disorder is prevented or,alternatively, delayed in its progression. Depending on the type of25934 aberrance, for example, a 25934, 25934 agonist or 25934 antagonistagent can be used for treating the subject. The appropriate agent can bedetermined based on screening assays described herein.

It is possible that some 25934 disorders can be caused, at least inpart, by an abnormal level of gene product, or by the presence of a geneproduct exhibiting abnormal activity. As such, the reduction in thelevel and/or activity of such gene products would bring about theamelioration of disorder symptoms.

In addition to the cardiovascular indications described above, the 25934molecules of the invention can be used to treat, prevent and/or diagnosedisorders involving the cells in which the 25934 mRNA is expressed(FIGS. 4-6). Accordingly, the molecules of the invention can be used totreat, present and/or diagnose neurological, reproductive (ovarian),renal, metabolic and hepatic disorders.

Disorders involving the brain include, but are not limited to, disordersinvolving neurons, and disorders involving glia, such as astrocytes,oligodendrocytes, ependymal cells, and microglia; cerebral edema, raisedintracranial pressure and herniation, and hydrocephalus; malformationsand developmental diseases, such as neural tube defects, forebrainanomalies, posterior fossa anomalies, and syringomyelia and hydromyelia;perinatal brain injury; cerebrovascular diseases, such as those relatedto hypoxia, ischemia, and infarction, including hypotension,hypoperfusion, and low-flow states—global cerebral ischemia and focalcerebral ischemia—infarction from obstruction of local blood supply,intracranial hemorrhage, including intracerebral (intraparenchymal)hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms, andvascular malformations, hypertensive cerebrovascular disease, includinglacunar infarcts, slit hemorrhages, and hypertensive encephalopathy;infections, such as acute meningitis, including acute pyogenic(bacterial) meningitis and acute aseptic (viral) meningitis, acute focalsuppurative infections, including brain abscess, subdural empyema, andextradural abscess, chronic bacterial meningoencephalitis, includingtuberculosis and mycobacterioses, neurosyphilis, and neuroborreliosis(Lyme disease), viral meningoencephalitis, including arthropod-borne(Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes simplexvirus Type 2, Varicalla-zoster virus (Herpes zoster), cytomegalovirus,poliomyelitis, rabies, and human immunodeficiency virus 1, includingHIV-1 meningoencephalitis (subacute encephalitis), vacuolar myelopathy,AIDS-associated myopathy, peripheral neuropathy, and AIDS in children,progressive multifocal leukoencephalopathy, subacute sclerosingpanencephalitis, fungal meningoencephalitis, other infectious diseasesof the nervous system; transmissible spongiform encephalopathies (priondiseases); demyelinating diseases, including multiple sclerosis,multiple sclerosis variants, acute disseminated encephalomyelitis andacute necrotizing hemorrhagic encephalomyelitis, and other diseases withdemyelination; degenerative diseases, such as degenerative diseasesaffecting the cerebral cortex, including Alzheimer disease and Pickdisease, degenerative diseases of basal ganglia and brain stem,including Parkinsonism, idiopathic Parkinson disease (paralysisagitans), progressive supranuclear palsy, corticobasal degenration,multiple system atrophy, including striatonigral degenration, Shy-Dragersyndrome, and olivopontocerebellar atrophy, and Huntington disease;spinocerebellar degenerations, including spinocerebellar ataxias,including Friedreich ataxia, and ataxia-telanglectasia, degenerativediseases affecting motor neurons, including amyotrophic lateralsclerosis (motor neuron disease), bulbospinal atrophy (Kennedysyndrome), and spinal muscular atrophy; inborn errors of metabolism,such as leukodystrophies, including Krabbe disease, metachromaticleukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, andCanavan disease, mitochondrial encephalomyopathies, including Leighdisease and other mitochondrial encephalomyopathies; toxic and acquiredmetabolic diseases, including vitamin deficiencies such as thiamine(vitamin B₁) deficiency and vitamin B₁₂ deficiency, neurologic sequelaeof metabolic disturbances, including hypoglycemia, hyperglycemia, andhepatic encephatopathy, toxic disorders, including carbon monoxide,methanol, ethanol, and radiation, including combined methotrexate andradiation-induced injury; tumors, such as gliomas, includingastrocytoma, including fibrillary (diffuse) astrocytoma and glioblastomamultiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, andbrain stem glioma, oligodendroglioma, and ependymoma and relatedparaventricular mass lesions, neuronal tumors, poorly differentiatedneoplasms, including medulloblastoma, other parenchymal tumors,including primary brain lymphoma, germ cell tumors, and pinealparenchymal tumors, meningiomas, metastatic tumors, paraneoplasticsyndromes, peripheral nerve sheath tumors, including schwannoma,neurofibroma, and malignant peripheral nerve sheath tumor (malignantschwannoma), and neurocutaneous syndromes (phakomatoses), includingneurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindaudisease.

Disorders involving the ovary include, for example, polycystic ovariandisease, Stein-leventhal syndrome, Pseudomyxoma peritonei and stromalhyperthecosis; ovarian tumors such as, tumors of coelomic epithelium,serous tumors, mucinous tumors, endometeriod tumors, clear celladenocarcinoma, cystadenofibroma, brenner tumor, surface epithelialtumors; germ cell tumors such as mature (benign) teratomas, monodermalteratomas, immature malignant teratomas, dysgerminoma, endodermal sinustumor, choriocarcinoma; sex cord-stomal tumors such as, granulosa-thecacell tumors, thecoma-fibromas, androblastomas, hill cell tumors, andgonadoblastoma; and metastatic tumors such as Krukenberg tumors.

Disorders involving the kidney include, but are not limited to,congenital anomalies including, but not limited to, cystic diseases ofthe kidney, that include but are not limited to, cystic renal dysplasia,autosomal dominant (adult) polycystic kidney disease, autosomalrecessive (childhood) polycystic kidney disease, and cystic diseases ofrenal medulla, which include, but are not limited to, medullary spongekidney, and nephronophthisis-uremic medullary cystic disease complex,acquired (dialysis-associated) cystic disease, such as simple cysts;glomerular diseases including pathologies of glomerular injury thatinclude, but are not limited to, in situ immune complex deposition, thatincludes, but is not limited to, anti-GBM nephritis, Heymann nephritis,and antibodies against planted antigens, circulating immune complexnephritis, antibodies to glomerular cells, cell-mediated immunity inglomerulonephritis, activation of alternative complement pathway,epithelial cell injury, and pathologies involving mediators ofglomerular injury including cellular and soluble mediators, acuteglomerulonephritis, such as acute proliferative (poststreptococcal,postinfectious) glomerulonephritis, including but not limited to,poststreptococcal glomerulonephritis and nonstreptococcal acuteglomerulonephritis, rapidly progressive (crescentic) glomerulonephritis,nephrotic syndrome, membranous glomerulonephritis (membranousnephropathy), minimal change disease (lipoid nephrosis), focal segmentalglomerulosclerosis, membranoproliferative glomerulonephritis, IgAnephropathy (Berger disease), focal proliferative and necrotizingglomerulonephritis (focal glomerulonephritis), hereditary nephritis,including but not limited to, Alport syndrome and thin membrane disease(benign familial hematuria), chronic glomerulonephritis, glomerularlesions associated with systemic disease, including but not limited to,systemic lupus erythematosus, Henoch-Schönlein purpura, bacterialendocarditis, diabetic glomerulosclerosis, amyloidosis, fibrillary andimmunotactoid glomerulonephritis, and other systemic disorders; diseasesaffecting tubules and interstitium, including acute tubular necrosis andtubulointerstitial nephritis, including but not limited to,pyelonephritis and urinary tract infection, acute pyelonephritis,chronic pyelonephritis and reflux nephropathy, and tubulointerstitialnephritis induced by drugs and toxins, including but not limited to,acute drug-induced interstitial nephritis, analgesic abuse nephropathy,nephropathy associated with nonsteroidal anti-inflammatory drugs, andother tubulointerstitial diseases including, but not limited to, uratenephropathy, hypercalcemia and nephrocalcinosis, and multiple myeloma;diseases of blood vessels including benign nephrosclerosis, malignanthypertension and accelerated nephrosclerosis, renal artery stenosis, andthrombotic microangiopathies including, but not limited to, classic(childhood) hemolytic-uremic syndrome, adult hemolytic-uremicsyndrome/thrombotic thrombocytopenic purpura, idiopathic HUS/TTP, andother vascular disorders including, but not limited to, atheroscleroticischemic renal disease, atheroembolic renal disease, sickle cell diseasenephropathy, diffuse cortical necrosis, and renal infarcts; urinarytract obstruction (obstructive uropathy); urolithiasis (renal calculi,stones); and tumors of the kidney including, but not limited to, benigntumors, such as renal papillary adenoma, renal fibroma or hamartoma(renomedullary interstitial cell tumor), angiomyolipoma, and oncocytoma,and malignant tumors, including renal cell carcinoma (hypemephroma,adenocarcinoma of kidney), which includes urothelial carcinomas of renalpelvis.

Disorders of the liver include, but are not limited to, disordersassociated with an accumulation in the liver of fibrous tissue, such asthat resulting from an imbalance between production and degradation ofthe extracellular matrix accompanied by the collapse and condensation ofpreexisting fibers. The methods described herein can be used to diagnoseor treat hepatocellular necrosis or injury induced by a wide variety ofagents including processes which disturb homeostasis, such as aninflammatory process, tissue damage resulting from toxic injury oraltered hepatic blood flow, and infections (e.g., bacterial, viral andparasitic). For example, the methods can be used for the early detectionof hepatic injury, such as portal hypertension or hepatic fibrosis. Inaddition, the methods can be employed to detect liver fibrosisattributed to inborn errors of metabolsim, for example, fibrosisresulting from a storage disorder such as Gaucher's disease (lipidabnormalities) or a glycogen storage disease, A1-antitrypsin deficiency;a disorder mediating the accumulation (e.g., storage) of an exogenoussubstance, for example, hemochromatosis (iron-overload syndrome) andcopper storage diseases (Wilson's disease), disorders resulting in theaccumulation of a toxic metabolite (e.g., tyrosinemia, fructosemia andgalactosemia) and peroxisomal disorders (e.g., Zellweger syndrome).Additionally, the methods described herein may be useful for the earlydetection and treatment of liver injury associated with theadministration of various chemicals or drugs, such as for example,methotrexate, isonizaid, oxyphenisatin, methyldopa, chlorpromazine,tolbutamide or alcohol, or which represents a hepatic manifestation of avascular disorder such as obstruction of either the intrahepatic orextrahepatic bile flow or an alteration in hepatic circulationresulting, for example, from chronic heart failure, veno-occlusivedisease, portal vein thrombosis or Budd-Chiari syndrome.

Additionally, 25934 molecules may play an important role in the etiologyof certain viral diseases, inducing but not limited to Hepatitis B,Heptitis C and Herpes Simplex Virus (HSV). Modulators of 25934 activitycould be used to control viral diseases. The modulators can be used inthe treatment and/or diagnosis of viral infected tissue orvirus-associated tissue fibrosis, especially liver and liver fibrosis.Also, 25934 modulators can be used in the treatment and/or diagnosis ofvirus-associated carcinoma, especially hepatocellular cancer.

Additionally, 25934 may play an important role in the regulation ofmetabolism. Diseases of metabolic imbalance include, but are not limitedto, obesity, anorexia nervosa, cachexia, lipid disorders diabetes.

As discussed, successful treatment of 25934 disorders can be broughtabout by techniques that serve to inhibit the expression or activity oftarget gene products. For example, compounds, e.g., an agent identifiedusing an assays described above, that proves to exhibit negativemodulatory activity, can be used in accordance with the invention toprevent and/or ameliorate symptoms of 25934 disorders. Such moleculescan include, but are not limited to peptides, phosphopeptides, smallorganic or inorganic molecules, or antibodies (including, for example,polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or singlechain antibodies, and Fab, F(ab′)₂ and FAb expression library fragments,scFV molecules, and epitope-binding fragments thereof).

Further, antisense and ribozyme molecules that inhibit expression of thetarget gene can also be used in accordance with the invention to reducethe level of target gene expression, thus effectively reducing the levelof target gene activity. Still further, triple helix molecules can beutilized in reducing the level of target gene activity. Antisense,ribozyme and triple helix molecules are discussed above.

It is possible that the use of antisense, ribozyme, and/or triple helixmolecules to reduce or inhibit mutant gene expression can also reduce orinhibit the transcription (triple helix) and/or translation (antisense,ribozyme) of mRNA produced by normal target gene alleles, such that theconcentration of normal target gene product present can be lower than isnecessary for a normal phenotype. In such cases, nucleic acid moleculesthat encode and express target gene polypeptides exhibiting normaltarget gene activity can be introduced into cells via gene therapymethod. Alternatively, in instances in that the target gene encodes anextracellular protein, it can be preferable to co-administer normaltarget gene protein into the cell or tissue in order to maintain therequisite level of cellular or tissue target gene activity.

Another method by which nucleic acid molecules may be utilized intreating or preventing a disease characterized by 25934 expression isthrough the use of aptamer molecules specific for 25934 protein.Aptamers are nucleic acid molecules having a tertiary structure whichpermits them to specifically bind to protein ligands (see, e.g.,Osborne, et al. Curr. Opin. Chem Biol. 1997, 1(1): 5-9; and Patel, D. J.Curr Opin Chem Biol 1997 June;1(1):32-46). Since nucleic acid moleculesmay in many cases be more conveniently introduced into target cells thantherapeutic protein molecules may be, aptamers offer a method by which25934 protein activity may be specifically decreased without theintroduction of drugs or other molecules which may have pluripotenteffects.

Antibodies can be generated that are both specific for target geneproduct and that reduce target gene product activity. Such antibodiesmay, therefore, by administered in instances whereby negative modulatorytechniques are appropriate for the treatment of 25934 disorders. For adescription of antibodies, see the Antibody section above.

In circumstances wherein injection of an animal or a human subject witha 25934 protein or epitope for stimulating antibody production isharmful to the subject, it is possible to generate an immune responseagainst 25934 through the use of anti-idiotypic antibodies (see, forexample, Herlyn, D. Ann Med 1999;31(1):66-78; andBhattacharya-Chatterjee, M., and Foon, K. A. Cancer Treat Res1998;94:51-68). If an anti-idiotypic antibody is introduced into amammal or human subject, it should stimulate the production ofanti-anti-idiotypic antibodies, which should be specific to the 25934protein. Vaccines directed to a disease characterized by 25934expression may also be generated in this fashion.

In instances where the target antigen is intracellular and wholeantibodies are used, internalizing antibodies may be preferred.Lipofectin or liposomes can be used to deliver the antibody or afragment of the Fab region that binds to the target antigen into cells.Where fragments of the antibody are used, the smallest inhibitoryfragment that binds to the target antigen is preferred. For example,peptides having an amino acid sequence corresponding to the Fv region ofthe antibody can be used. Alternatively, single chain neutralizingantibodies that bind to intracellular target antigens can also beadministered. Such single chain antibodies can be administered, forexample, by expressing nucleotide sequences encoding single-chainantibodies within the target cell population (see e.g., Marasco et al.(1993, Proc. Natl. Acad. Sci. USA 90:7889-7893).

The identified compounds that inhibit target gene expression, synthesisand/or activity can be administered to a patient at therapeuticallyeffective doses to prevent, treat or ameliorate 25934 disorders. Atherapeutically effective dose refers to that amount of the compoundsufficient to result in amelioration of symptoms of the disorders.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

Another example of determination of effective dose for an individual isthe ability to directly assay levels of “free” and “bound” compound inthe serum of the test subject. Such assays may utilize antibody mimicsand/or “biosensors” that have been created through molecular imprintingtechniques. The compound which is able to modulate 25934 activity isused as a template, or “imprinting molecule”, to spatially organizepolymerizable monomers prior to their polymerization with catalyticreagents. The subsequent removal of the imprinted molecule leaves apolymer matrix which contains a repeated “negative image” of thecompound and is able to selectively rebind the molecule under biologicalassay conditions. A detailed review of this technique can be seen inAnsell, R. J. et al (1996) Current Opinion in Biotechnology 7:89-94 andin Shea, K. J. (1994) Trends in Polymer Science 2:166-173. Such“imprinted” affinity matrixes are amenable to ligand-binding assays,whereby the immobilized monoclonal antibody component is replaced by anappropriately imprinted matrix. An example of the use of such matrixesin this way can be seen in Vlatakis, G. et al (1993) Nature 361:645-647.Through the use of isotope-labeling, the “free” concentration ofcompound which modulates the expression or activity of 25934 can bereadily monitored and used in calculations of IC₅₀.

Such “imprinted” affinity matrixes can also be designed to includefluorescent groups whose photon-emitting properties measurably changeupon local and selective binding of target compound. These changes canbe readily assayed in real time using appropriate fiberoptic devices, inturn allowing the dose in a test subject to be quickly optimized basedon its individual IC₅₀. An rudimentary example of such a “biosensor” isdiscussed in Kriz, D. et al (1995) Analytical Chemistry 67:2142-2144.

Another aspect of the invention pertains to methods of modulating 25934expression or activity for therapeutic purposes. Accordingly, in anexemplary embodiment, the modulatory method of the invention involvescontacting a cell with a 25934 or agent that modulates one or more ofthe activities of 25934 protein activity associated with the cell. Anagent that modulates 25934 protein activity can be an agent as describedherein, such as a nucleic acid or a protein, a naturally-occurringtarget molecule of a 25934 protein (e.g., a 25934 substrate orreceptor), a 25934 antibody, a 25934 agonist or antagonist, apeptidomimetic of a 25934 agonist or antagonist, or other smallmolecule.

In one embodiment, the agent stimulates one or 25934 activities.Examples of such stimulatory agents include active 25934 protein and anucleic acid molecule encoding 25934. In another embodiment, the agentinhibits one or more 25934 activities. Examples of such inhibitoryagents include antisense 25934 nucleic acid molecules, anti25934antibodies, and 25934 inhibitors. These modulatory methods can beperformed in vitro (e.g., by culturing the cell with the agent) or,alternatively, in vivo (e.g., by administering the agent to a subject).As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant or unwanted expression or activity of a 25934 protein ornucleic acid molecule. In one embodiment, the method involvesadministering an agent (e.g., an agent identified by a screening assaydescribed herein), or combination of agents that modulates (e.g.,upregulates or downregulates) 25934 expression or activity. In anotherembodiment, the method involves administering a 25934 protein or nucleicacid molecule as therapy to compensate for reduced, aberrant, orunwanted 25934 expression or activity.

Stimulation of 25934 activity is desirable in situations in which 25934is abnormally downregulated and/or in which increased 25934 activity islikely to have a beneficial effect. For example, stimulation of 25934activity is desirable in situations in which a 25934 is downregulatedand/or in which increased 25934 activity is likely to have a beneficialeffect. Likewise, inhibition of 25934 activity is desirable insituations in which 25934 is abnormally upregulated and/or in whichdecreased 25934 activity is likely to have a beneficial effect.

Pharmacogenomics

The 25934 molecules of the present invention, as well as agents, ormodulators which have a stimulatory or inhibitory effect on 25934activity (e.g., 25934 gene expression) as identified by a screeningassay described herein can be administered to individuals to treat(prophylactically or therapeutically) 25934 associated disorders (e.g.,diabetes, obesity, hypertension, cancer, developmental disorders, immunedisorders and neurological and heart diseases) associated with aberrantor unwanted 25934 activity. In conjunction with such treatment,pharmacogenomics (i.e., the study of the relationship between anindividual's genotype and that individual's response to a foreigncompound or drug) may be considered. Differences in metabolism oftherapeutics can lead to severe toxicity or therapeutic failure byaltering the relation between dose and blood concentration of thepharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a 25934 molecule or 25934modulator as well as tailoring the dosage and/or therapeutic regimen oftreatment with a 25934 molecule or 25934 modulator.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, for example, Eichelbaum, M. et al.(1996) Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditionstransmitted as a single factor altering the way drugs act on the body(altered drug action) or genetic conditions transmitted as singlefactors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raregenetic defects or as naturally-occurring polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association”, relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

Alternatively, a method termed the “candidate gene approach”, can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drug's target is known (e.g., a 25934protein of the present invention), all common variants of that gene canbe fairly easily identified in the population and it can be determinedif having one version of the gene versus another is associated with aparticular drug response.

Alternatively, a method termed the “gene expression profiling”, can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug (e.g., a 25934 moleculeor 25934 modulator of the present invention) can give an indicationwhether gene pathways related to toxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment of an individual.This knowledge, when applied to dosing or drug selection, can avoidadverse reactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a 25934 molecule or25934 modulator, such as a modulator identified by one of the exemplaryscreening assays described herein.

The present invention further provides methods for identifying newagents, or combinations, that are based on identifying agents thatmodulate the activity of one or more of the gene products encoded by oneor more of the 25934 genes of the present invention, wherein theseproducts may be associated with resistance of the cells to a therapeuticagent. Specifically, the activity of the proteins encoded by the 25934genes of the present invention can be used as a basis for identifyingagents for overcoming agent resistance. By blocking the activity of oneor more of the resistance proteins, target cells, e.g., adipocyte cells,will become sensitive to treatment with an agent that the unmodifiedtarget cells were resistant to.

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of a 25934 protein can be applied in clinical trials. Forexample, the effectiveness of an agent determined by a screening assayas described herein to increase 25934 gene expression, protein levels,or upregulate 25934 activity, can be monitored in clinical trials ofsubjects exhibiting decreased 25934 gene expression, protein levels, ordownregulated 25934 activity. Alternatively, the effectiveness of anagent determined by a screening assay to decrease 25934 gene expression,protein levels, or downregulate 25934 activity, can be monitored inclinical trials of subjects exhibiting increased 25934 gene expression,protein levels, or upregulated 25934 activity. In such clinical trials,the expression or activity of a 25934 gene, and preferably, other genesthat have been implicated in, for example, a 25934-associated disordercan be used as a “read out” or markers of the phenotype of a particularcell.

Other Embodiments

In another aspect, the invention features, a method of analyzing aplurality of capture probes. The method can be used, e.g., to analyzegene expression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the plurality,and each address of the plurality having a unique capture probe, e.g., anucleic acid or peptide sequence; contacting the array with a 25934,preferably purified, nucleic acid, preferably purified, polypeptide,preferably purified, or antibody, and thereby evaluating the pluralityof capture probes. Binding, e.g., in the case of a nucleic acid,hybridization with a capture probe at an address of the plurality, isdetected, e.g., by signal generated from a label attached to the 25934nucleic acid, polypeptide, or antibody.

The capture probes can be a set of nucleic acids from a selected sample,e.g., a sample of nucleic acids derived from a control or non-stimulatedtissue or cell.

The method can include contacting the 25934 nucleic acid, polypeptide,or antibody with a first array having a plurality of capture probes anda second array having a different plurality of capture probes. Theresults of each hybridization can be compared, e.g., to analyzedifferences in expression between a first and second sample. The firstplurality of capture probes can be from a control sample, e.g., a wildtype, normal, or non-diseased, non-stimulated, sample, e.g., abiological fluid, tissue, or cell sample. The second plurality ofcapture probes can be from an experimental sample, e.g., a mutant type,at risk, disease-state or disorder-state, or stimulated, sample, e.g., abiological fluid, tissue, or cell sample.

The plurality of capture probes can be a plurality of nucleic acidprobes each of which specifically hybridizes, with an allele of 25934.Such methods can be used to diagnose a subject, e.g., to evaluate riskfor a disease or disorder, to evaluate suitability of a selectedtreatment for a subject, to evaluate whether a subject has a disease ordisorder. 25934 is associated with regulating membrane fluidity, thus itis useful for evaluating a number of diseases such as diabetes, obesity,hypertension, cancer, developmental disorders, immune disorders andneurological and heart diseases.

The method can be used to detect SNPs, as described above.

In another aspect, the invention features, a method of analyzing aplurality of probes. The method is useful, e.g., for analyzing geneexpression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the pluralityhaving a unique capture probe, e.g., wherein the capture probes are froma cell or subject which express 25934 or from a cell or subject in whicha 25934 mediated response has been elicited, e.g., by contact of thecell with 25934 nucleic acid or protein, or administration to the cellor subject 25934 nucleic acid or protein; contacting the array with oneor more inquiry probe, wherein an inquiry probe can be a nucleic acid,polypeptide, or antibody (which is preferably other than 25934 nucleicacid, polypeptide, or antibody); providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the plurality,and each address of the plurality having a unique capture probe, e.g.,wherein the capture probes are from a cell or subject which does notexpress 25934 (or does not express as highly as in the case of the 25934positive plurality of capture probes) or from a cell or subject which inwhich a 25934 mediated response has not been elicited (or has beenelicited to a lesser extent than in the first sample); contacting thearray with one or more inquiry probes (which is preferably other than a25934 nucleic acid, polypeptide, or antibody), and thereby evaluatingthe plurality of capture probes. Binding, e.g., in the case of a nucleicacid, hybridization with a capture probe at an address of the plurality,is detected, e.g., by signal generated from a label attached to thenucleic acid, polypeptide, or antibody.

In another aspect, the invention features, a method of analyzing 25934,e.g., analyzing structure, function, or relatedness to other nucleicacid or amino acid sequences. The method includes: providing a 25934nucleic acid or amino acid sequence; comparing the 25934 sequence withone or more preferably a plurality of sequences from a collection ofsequences, e.g., a nucleic acid or protein sequence database; to therebyanalyze 25934.

Preferred databases include Genbank. The method can include evaluatingthe sequence identity between a 25934 sequence and a database sequence.The method can be performed by accessing the database at a second site,e.g., over the internet.

In another aspect, the invention features, a set of oligonucleotides,useful, e.g., for identifying SNP's, or identifying specific alleles of25934. The set includes a plurality of oligonucleotides, each of whichhas a different nucleotide at an interrogation position, e.g., an SNP orthe site of a mutation. In a preferred embodiment, the oligonucleotidesof the plurality identical in sequence with one another (except fordifferences in length). The oligonucleotides can be provided withdiferential labels, such that an oligonucleotides which hybridizes toone allele provides a signal that is distinguishable from anoligonucleotides which hybridizes to a second allele.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are incorporated herein by reference.

EXAMPLES Example 1 Identification and Characterization of Human 25934cDNA

The human 25934 sequence (FIGS. 1A-B; SEQ ID NO:1), which isapproximately 1512 nucleotides long including untranslated regions,contains a predicted methionine-initiated coding sequence of about 990nucleotides (nucleotides 403 to 1392 of SEQ ID NO:1; SEQ ID NO:3). Thecoding sequence encodes a 330 amino acid protein (SEQ ID NO:2).

Example 2 Tissue Distribution of 25934 mRNA

Endogenous human 25934 gene expression was determined using thePerkin-Elmer/ABI 7700 Sequence Detection System which employs TaqMantechnology. Briefly, TaqMan technology relies on standard RT-PCR withthe addition of a third gene-specific oligonucleotide (referred to as aprobe) which has a fluorescent dye coupled to its 5′ end (typically6-FAM) and a quenching dye at the 3′ end (typically TAMRA). When thefluorescently tagged oligonucleotide is intact, the fluorescent signalfrom the 5′ dye is quenched. As PCR proceeds, the 5′ to 3′ nucleolyticactivity of Taq polymerase digests the labeled primer, producing a freenucleotide labeled with 6-FAM, which is now detected as a fluorescentsignal. The PCR cycle where fluorescence is first released and detectedis directly proportional to the starting amount of the gene of interestin the test sample, thus providing a way of quantitating the initialtemplate concentration. Samples can be internally controlled by theaddition of a second set of primers/probe specific for a housekeepinggene such as GAPDH which has been labeled with a different fluorophoreon the 5′ end (typically VIC).

To determine the level of 25934 in various human tissues a primer/probeset was designed using Primer Express (Perkin-Elmer) software andprimary cDNA sequence information. Total RNA was prepared from a seriesof human tissues using an RNeasy kit from Qiagen. First strand cDNA wasprepared from 1 μg total RNA using an oligo-dT primer and Superscript IIreverse transcriptase (Gibco/BRL). cDNA obtained from approximately 50ng total RNA was used per TaqMan reaction.

25934 mRNA levels were analyzed in a variety of samples of isolated fromthe human fetal heart, spinal cord, brain (cortex, hypothalamus, glialcells), ovary, kidney, liver, endothelial cells and smooth muscle cells(SMC). The highest relative 25934 mRNA expression, i.e., greater than200 relative units, was observed in spinal cord, brain and ovary (FIG.4). High level mRNA expression, i.e., greater than 100 relative unitswas observed in the kidney, endothelial cells and human umbilical veinendothelial cells (HUVEC) (FIG. 4). Expression in liver (a target organfor 25934) was positive but lower relative to other tissues.

The relative 25934 mRNA expression as determined by TaqMan assays onmRNA derived a panel of human liver tissues is shown in FIG. 5. FIG. 6shows a comparison of the expression of 25934 mRNA and stearoyl CoAdesaturase (SCD) mRNA in a panel of human liver. Expression of 25934mRNA in human liver is equivalent to the relative expression of theknown SCD gene.

FIG. 7 shows the inhibition of 25934 mRNA expression in the marmosetanimal model. Niacin treatment in the marmoset model results insignificant repression of 25934 in the liver.

Northern blot hybridizations with various RNA samples can be performedunder standard conditions and washed under stringent conditions, i.e.,0.2×SSC at 65° C. A DNA probe corresponding to all or a portion of the25934 cDNA (SEQ ID NO:1) is used. The DNA is radioactively labeled with³²P-dCTP using the Prime-It Kit (Stratagene, La Jolla, Calif.) accordingto the instructions of the supplier. Filters containing mRNA from mousehematopoietic and endocrine tissues, and cancer cell lines (Clontech,Palo Alto, Calif.) is probed in ExpressHyb hybridization solution(Clontech) and washed at high stringency according to manufacturer'srecommendations.

Example 3 Recombinant Expression of 25934 in Bacterial Cells

In this example, 25934 is expressed as a recombinantglutathione-S-transferase (GST) fusion polypeptide in E. coli and thefusion polypeptide is isolated and characterized. Specifically, 25934 isfused to GST and this fusion polypeptide is expressed in E. coli, e.g.,strain PEB199. Expression of the GST-25934 fusion protein in PEB 199 isinduced with IPTG. The recombinant fusion polypeptide is purified fromcrude bacterial lysates of the induced PEB 199 strain by affinitychromatography on glutathione beads. Using polyacrylamide gelelectrophoretic analysis of the polypeptide purified from the bacteriallysates, the molecular weight of the resultant fusion polypeptide isdetermined.

Example 4 Expression of Recombinant 25934 Protein in COS Cells

To express the 25934 gene in COS cells, the pcDNA/Amp vector byInvitrogen Corporation (San Diego, Calif.) is used. This vector containsan SV40 origin of replication, an ampicillin resistance gene, an E. colireplication origin, a CMV promoter followed by a polylinker region, andan SV40 intron and polyadenylation site. A DNA fragment encoding theentire 25934 protein and an HA tag (Wilson et al. (1984) Cell 37:767) ora FLAG tag fused in-frame to its 3′ end of the fragment is cloned intothe polylinker region of the vector, thereby placing the expression ofthe recombinant protein under the control of the CMV promoter.

To construct the plasmid, the 25934 DNA sequence is amplified by PCRusing two primers. The 5′ primer contains the restriction site ofinterest followed by approximately twenty nucleotides of the 25934coding sequence starting from the initiation codon; the 3′ end sequencecontains complementary sequences to the other restriction site ofinterest, a translation stop codon, the HA tag or FLAG tag and the last20 nucleotides of the 25934 coding sequence. The PCR amplified fragmentand the pCDNA/Amp vector are digested with the appropriate restrictionenzymes and the vector is dephosphorylated using the CIAP enzyme (NewEngland Biolabs, Beverly, Mass.). Preferably the two restriction siteschosen are different so that the 25934 gene is inserted in the correctorientation. The ligation mixture is transformed into E. coli cells(strains HB101, DH5α, SURE, available from Stratagene Cloning Systems,La Jolla, Calif., can be used), the transformed culture is plated onampicillin media plates, and resistant colonies are selected. PlasmidDNA is isolated from transformants and examined by restriction analysisfor the presence of the correct fragment.

COS cells are subsequently transfected with the 25934-pcDNA/Amp plasmidDNA using the calcium phosphate or calcium chloride co-precipitationmethods, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Other suitable methods for transfecting host cells canbe found in Sambrook, J., Fritsh, E. F., and Maniatis, T. MolecularCloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Theexpression of the 25934 polypeptide is detected by radiolabelling(³⁵S-methionine or ³⁵S-cysteine available from NEN, Boston, Mass., canbe used) and immunoprecipitation (Harlow, E. and Lane, D. Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1988) using an HA specific monoclonal antibody. Briefly,the cells are labeled for 8 hours with ³⁵S-methionine (or ³⁵S-cysteine).The culture media are then collected and the cells are lysed usingdetergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50mM Tris, pH 7.5). Both the cell lysate and the culture media areprecipitated with an HA specific monoclonal antibody. Precipitatedpolypeptides are then analyzed by SDS-PAGE.

Alternatively, DNA containing the 25934 coding sequence is cloneddirectly into the polylinker of the pcDNA/Amp vector using theappropriate restriction sites. The resulting plasmid is transfected intoCOS cells in the manner described above, and the expression of the 25934polypeptide is detected by radiolabelling and immunoprecipitation usinga 25934 specific monoclonal antibody.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

9 1 1512 DNA Homo sapiens CDS (342)...(1331) 1 ccacgcgtcc ggactagttccatttccaca gctcctcctc cccggccgcg cgcccctccc 60 gccccgcgcg cgcctcctctttctcgcggc cgagttcagc ccgggcagcc atatggggga 120 tacgccagca acagacgccggccgccaaga tctgcatccc taggccacgc taagaccctg 180 gggaagagcg caggagcccgggagaagggc tggaaggagg ggactggacg tgcggagaat 240 tcccccctaa aaggcagaagcccccgcccc caccctcgag ctccgctcgg gcagagcgcc 300 tgcctgcctg ccgctgctgcgggcgcccac ctcgcccagc c atg cca ggc ccg gcc 356 Met Pro Gly Pro Ala 1 5acc gac gcg ggg aag atc cct ttc tgc gac gcc aag gaa gaa atc cgt 404 ThrAsp Ala Gly Lys Ile Pro Phe Cys Asp Ala Lys Glu Glu Ile Arg 10 15 20 gccggg ctc gaa agc tct gag ggc ggc ggc ggc ccg gag agg cca ggc 452 Ala GlyLeu Glu Ser Ser Glu Gly Gly Gly Gly Pro Glu Arg Pro Gly 25 30 35 gcg cgcggg cag cgg cag aac atc gtc tgg agg aat gtc gtc ctg atg 500 Ala Arg GlyGln Arg Gln Asn Ile Val Trp Arg Asn Val Val Leu Met 40 45 50 agc ttg ctccac ttg ggg gcc gtg tac tcc ctg gtg ctc atc ccc aaa 548 Ser Leu Leu HisLeu Gly Ala Val Tyr Ser Leu Val Leu Ile Pro Lys 55 60 65 gcc aag cca ctcact ctg ctc tgg gcc tac ttc tgc ttc ctc ctg gcc 596 Ala Lys Pro Leu ThrLeu Leu Trp Ala Tyr Phe Cys Phe Leu Leu Ala 70 75 80 85 gct ctg ggt gtgaca gct ggt gcc cat cgc ttg tgg agc cac agg tcc 644 Ala Leu Gly Val ThrAla Gly Ala His Arg Leu Trp Ser His Arg Ser 90 95 100 tac cgg gcc aagctg cct ctg agg ata ttt ctg gct gtc gcc aac tcc 692 Tyr Arg Ala Lys LeuPro Leu Arg Ile Phe Leu Ala Val Ala Asn Ser 105 110 115 atg gct ttc cagaat gac atc ttc gag tgg tcc agg gac cac cga gcc 740 Met Ala Phe Gln AsnAsp Ile Phe Glu Trp Ser Arg Asp His Arg Ala 120 125 130 cac cac aag tactca gag acg gat gct gac ccc cac aat gcc cgc cgg 788 His His Lys Tyr SerGlu Thr Asp Ala Asp Pro His Asn Ala Arg Arg 135 140 145 ggc ttc ttc ttctcc cat att ggg tgg ctg ttt gtt cgc aag cat cga 836 Gly Phe Phe Phe SerHis Ile Gly Trp Leu Phe Val Arg Lys His Arg 150 155 160 165 gat gtt attgag aag ggg aga aag ctt gac gtc act gac ctg ctt gct 884 Asp Val Ile GluLys Gly Arg Lys Leu Asp Val Thr Asp Leu Leu Ala 170 175 180 gat cct gtggtc cgg atc cag aga aag tac tat aag atc tcc gtg gtg 932 Asp Pro Val ValArg Ile Gln Arg Lys Tyr Tyr Lys Ile Ser Val Val 185 190 195 ctc atg tgcttt gtg gtc ccc acg ctg gtg ccc tgg tac atc tgg gga 980 Leu Met Cys PheVal Val Pro Thr Leu Val Pro Trp Tyr Ile Trp Gly 200 205 210 gag agt ctgtgg aat tcc tac ttc ttg gcc tct att ctc cgc tat acc 1028 Glu Ser Leu TrpAsn Ser Tyr Phe Leu Ala Ser Ile Leu Arg Tyr Thr 215 220 225 atc tca ctcaac atc agc tgg ctg gtc aac agc gcc gcc cac atg tat 1076 Ile Ser Leu AsnIle Ser Trp Leu Val Asn Ser Ala Ala His Met Tyr 230 235 240 245 gga aaccgg ccc tat gac aag cac atc agc cct cgg cag aac cca ctc 1124 Gly Asn ArgPro Tyr Asp Lys His Ile Ser Pro Arg Gln Asn Pro Leu 250 255 260 gtc gctctg ggt gcc att ggt gaa ggc ttc cat aat tac cat cac acc 1172 Val Ala LeuGly Ala Ile Gly Glu Gly Phe His Asn Tyr His His Thr 265 270 275 ttt cccttt gac tac tct gcg agt gaa ttt ggc tta aat ttt aac cca 1220 Phe Pro PheAsp Tyr Ser Ala Ser Glu Phe Gly Leu Asn Phe Asn Pro 280 285 290 acc acctgg ttc att gat ttc atg tgc tgg ctg ggg ctg gcc act gac 1268 Thr Thr TrpPhe Ile Asp Phe Met Cys Trp Leu Gly Leu Ala Thr Asp 295 300 305 cgc aaacgg gca acc aag ccg atg atc gag gcc cgg aag gcc agg act 1316 Arg Lys ArgAla Thr Lys Pro Met Ile Glu Ala Arg Lys Ala Arg Thr 310 315 320 325 ggagac agc agt gct tgaacttgga acagccatcc cacatgtctg ccgttgcaac 1371 Gly AspSer Ser Ala 330 ctcggttcat ggctttggtt acaatagctc tcttgtacat tggatcgtgggagggggcag 1431 agggtgggga aggaacgagt caatgtggtt tgggaatgtt tttgtttatctcaaaataat 1491 gttgaaatac aattatcaat g 1512 2 330 PRT Homo sapiens 2Met Pro Gly Pro Ala Thr Asp Ala Gly Lys Ile Pro Phe Cys Asp Ala 1 5 1015 Lys Glu Glu Ile Arg Ala Gly Leu Glu Ser Ser Glu Gly Gly Gly Gly 20 2530 Pro Glu Arg Pro Gly Ala Arg Gly Gln Arg Gln Asn Ile Val Trp Arg 35 4045 Asn Val Val Leu Met Ser Leu Leu His Leu Gly Ala Val Tyr Ser Leu 50 5560 Val Leu Ile Pro Lys Ala Lys Pro Leu Thr Leu Leu Trp Ala Tyr Phe 65 7075 80 Cys Phe Leu Leu Ala Ala Leu Gly Val Thr Ala Gly Ala His Arg Leu 8590 95 Trp Ser His Arg Ser Tyr Arg Ala Lys Leu Pro Leu Arg Ile Phe Leu100 105 110 Ala Val Ala Asn Ser Met Ala Phe Gln Asn Asp Ile Phe Glu TrpSer 115 120 125 Arg Asp His Arg Ala His His Lys Tyr Ser Glu Thr Asp AlaAsp Pro 130 135 140 His Asn Ala Arg Arg Gly Phe Phe Phe Ser His Ile GlyTrp Leu Phe 145 150 155 160 Val Arg Lys His Arg Asp Val Ile Glu Lys GlyArg Lys Leu Asp Val 165 170 175 Thr Asp Leu Leu Ala Asp Pro Val Val ArgIle Gln Arg Lys Tyr Tyr 180 185 190 Lys Ile Ser Val Val Leu Met Cys PheVal Val Pro Thr Leu Val Pro 195 200 205 Trp Tyr Ile Trp Gly Glu Ser LeuTrp Asn Ser Tyr Phe Leu Ala Ser 210 215 220 Ile Leu Arg Tyr Thr Ile SerLeu Asn Ile Ser Trp Leu Val Asn Ser 225 230 235 240 Ala Ala His Met TyrGly Asn Arg Pro Tyr Asp Lys His Ile Ser Pro 245 250 255 Arg Gln Asn ProLeu Val Ala Leu Gly Ala Ile Gly Glu Gly Phe His 260 265 270 Asn Tyr HisHis Thr Phe Pro Phe Asp Tyr Ser Ala Ser Glu Phe Gly 275 280 285 Leu AsnPhe Asn Pro Thr Thr Trp Phe Ile Asp Phe Met Cys Trp Leu 290 295 300 GlyLeu Ala Thr Asp Arg Lys Arg Ala Thr Lys Pro Met Ile Glu Ala 305 310 315320 Arg Lys Ala Arg Thr Gly Asp Ser Ser Ala 325 330 3 990 DNA Homosapiens 3 atgccaggcc cggccaccga cgcggggaag atccctttct gcgacgccaaggaagaaatc 60 cgtgccgggc tcgaaagctc tgagggcggc ggcggcccgg agaggccaggcgcgcgcggg 120 cagcggcaga acatcgtctg gaggaatgtc gtcctgatga gcttgctccacttgggggcc 180 gtgtactccc tggtgctcat ccccaaagcc aagccactca ctctgctctgggcctacttc 240 tgcttcctcc tggccgctct gggtgtgaca gctggtgccc atcgcttgtggagccacagg 300 tcctaccggg ccaagctgcc tctgaggata tttctggctg tcgccaactccatggctttc 360 cagaatgaca tcttcgagtg gtccagggac caccgagccc accacaagtactcagagacg 420 gatgctgacc cccacaatgc ccgccggggc ttcttcttct cccatattgggtggctgttt 480 gttcgcaagc atcgagatgt tattgagaag gggagaaagc ttgacgtcactgacctgctt 540 gctgatcctg tggtccggat ccagagaaag tactataaga tctccgtggtgctcatgtgc 600 tttgtggtcc ccacgctggt gccctggtac atctggggag agagtctgtggaattcctac 660 ttcttggcct ctattctccg ctataccatc tcactcaaca tcagctggctggtcaacagc 720 gccgcccaca tgtatggaaa ccggccctat gacaagcaca tcagccctcggcagaaccca 780 ctcgtcgctc tgggtgccat tggtgaaggc ttccataatt accatcacacctttcccttt 840 gactactctg cgagtgaatt tggcttaaat tttaacccaa ccacctggttcattgatttc 900 atgtgctggc tggggctggc cactgaccgc aaacgggcaa ccaagccgatgatcgaggcc 960 cggaaggcca ggactggaga cagcagtgct 990 4 248 PRT ArtificialSequence consensus sequence 4 Ile Leu Leu Gly Ala Leu His Leu Gly AlaLeu Tyr Leu Leu Ala Leu 1 5 10 15 Leu Pro Thr Glu Leu Lys Trp Lys ThrVal Ile Val Ala Leu Leu Leu 20 25 30 Tyr Val Ile Thr Gly Gly Leu Gly IleThr Ala Gly Tyr His Arg Leu 35 40 45 Trp Ser His Arg Ser Tyr Lys Ala LysLeu Pro Leu Arg Ile Phe Leu 50 55 60 Ala Ile Phe Gly Thr Leu Ala Val GlnGly Ser Ile Tyr Glu Trp Ala 65 70 75 80 Arg Asp His Arg Ala His His LysTyr Ser Asp Thr Asp Ala Asp Pro 85 90 95 His Asp Ala Asn Arg Gly Phe PhePhe Ser His Val Gly Trp Leu Leu 100 105 110 Val Lys Lys His Pro Ala ValLys Glu Lys Gly Lys Lys Leu Asp Leu 115 120 125 Ser Asp Leu Lys Ala AspPro Val Val Arg Phe Gln His Arg Tyr Tyr 130 135 140 Ile Pro Leu Met ValLeu Met Gly Phe Ile Leu Pro Thr Leu Val Pro 145 150 155 160 Gly Tyr LeuTrp Gly Glu Thr Phe Trp Gly Gly Phe Val Trp Ala Gly 165 170 175 Phe LeuArg Leu Val Phe Val Leu His Ala Thr Trp Cys Val Asn Ser 180 185 190 AlaAla His Lys Phe Gly Tyr Arg Pro Tyr Asp Ser Arg Ile Thr Pro 195 200 205Arg Asn Asn Trp Leu Val Ala Leu Val Thr Phe Gly Glu Gly Trp His 210 215220 Asn Phe His His Thr Phe Pro Tyr Asp Tyr Arg Asn Ala Glu Lys Trp 225230 235 240 Lys Trp Glu Tyr Asp Leu Thr Lys 245 5 248 PRT ArtificialSequence conserved binding motif 5 His Xaa Xaa Xaa Xaa His Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa His 35 40 45 Xaa Xaa Xaa His His Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135 140 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 145 150 155 160 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 165 170 175 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 180 185 190Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 195 200205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 210215 220 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa225 230 235 240 Xaa Xaa His Xaa Xaa Xaa His His 245 6 15 PRT ArtificialSequence motif 6 Gly Glu Xaa Xaa His Asn Xaa His His Xaa Phe Pro Xaa AspTyr 1 5 10 15 7 359 PRT Homo sapiens 7 Met Pro Ala His Leu Leu Gln AspAsp Ile Ser Ser Ser Tyr Thr Thr 1 5 10 15 Thr Thr Thr Ile Thr Ala ProPro Ser Arg Val Leu Gln Asn Gly Gly 20 25 30 Asp Lys Leu Glu Thr Met ProLeu Tyr Leu Glu Asp Asp Ile Arg Pro 35 40 45 Asp Ile Lys Asp Asp Ile TyrAsp Pro Thr Tyr Lys Asp Lys Glu Gly 50 55 60 Pro Ser Pro Lys Val Glu TyrVal Trp Arg Asn Ile Ile Leu Met Ser 65 70 75 80 Leu Leu His Leu Gly AlaLeu Tyr Gly Ile Thr Leu Ile Pro Thr Cys 85 90 95 Lys Phe Tyr Thr Trp LeuTrp Gly Val Phe Tyr Tyr Phe Val Ser Ala 100 105 110 Leu Gly Ile Thr AlaGly Ala His Arg Leu Trp Ser His Arg Ser Tyr 115 120 125 Lys Ala Arg LeuPro Leu Arg Leu Phe Leu Ile Ile Ala Asn Thr Met 130 135 140 Ala Phe GlnAsn Asp Val Tyr Glu Trp Ala Arg Asp His Arg Ala His 145 150 155 160 HisLys Phe Ser Glu Thr His Ala Asp Pro His Asn Ser Arg Arg Gly 165 170 175Phe Phe Phe Ser His Val Gly Trp Leu Leu Val Arg Lys His Pro Ala 180 185190 Val Lys Glu Lys Gly Ser Thr Leu Asp Leu Ser Asp Leu Glu Ala Glu 195200 205 Lys Leu Val Met Phe Gln Arg Arg Tyr Tyr Lys Pro Gly Leu Leu Leu210 215 220 Met Cys Phe Ile Leu Pro Thr Leu Val Pro Trp Tyr Phe Trp GlyGlu 225 230 235 240 Thr Phe Gln Asn Ser Val Phe Val Ala Thr Phe Leu ArgTyr Ala Val 245 250 255 Val Leu Asn Ala Thr Trp Leu Val Asn Ser Ala AlaHis Leu Phe Gly 260 265 270 Tyr Arg Pro Tyr Asp Lys Asn Ile Ser Pro ArgGlu Asn Ile Leu Val 275 280 285 Ser Leu Gly Ala Val Gly Glu Gly Phe HisAsn Tyr His His Ser Phe 290 295 300 Pro Tyr Asp Tyr Ser Ala Ser Glu TyrArg Trp His Ile Asn Phe Thr 305 310 315 320 Thr Phe Phe Ile Asp Cys MetAla Ala Leu Gly Leu Ala Tyr Asp Arg 325 330 335 Lys Lys Val Ser Lys AlaAla Ile Leu Ala Arg Ile Lys Arg Thr Gly 340 345 350 Asp Gly Asn Tyr LysSer Gly 355 8 358 PRT Rattus norvegicus 8 Met Pro Ala His Met Leu GlnGlu Ile Ser Ser Ser Tyr Thr Thr Thr 1 5 10 15 Thr Thr Ile Thr Glu ProPro Ser Gly Asn Leu Gln Asn Gly Arg Glu 20 25 30 Lys Met Lys Lys Val ProLeu Tyr Leu Glu Glu Asp Ile Arg Pro Glu 35 40 45 Met Arg Glu Asp Ile HisAsp Pro Ser Tyr Gln Asp Glu Glu Gly Pro 50 55 60 Pro Pro Lys Leu Glu TyrVal Trp Arg Asn Ile Ile Leu Met Ala Leu 65 70 75 80 Leu His Val Gly AlaLeu Tyr Gly Ile Thr Leu Ile Pro Ser Ser Lys 85 90 95 Val Tyr Thr Leu LeuTrp Gly Ile Phe Tyr Tyr Leu Ile Ser Ala Leu 100 105 110 Gly Ile Thr AlaGly Ala His Arg Leu Trp Ser His Arg Thr Tyr Lys 115 120 125 Ala Arg LeuPro Leu Arg Ile Phe Leu Ile Ile Ala Asn Thr Met Ala 130 135 140 Phe GlnAsn Asp Val Tyr Glu Trp Ala Arg Asp His Arg Ala His His 145 150 155 160Lys Phe Ser Glu Thr His Ala Asp Pro His Asn Ser Arg Arg Gly Phe 165 170175 Phe Phe Ser His Val Gly Trp Leu Leu Val Arg Lys His Pro Ala Val 180185 190 Lys Glu Lys Gly Gly Lys Leu Asp Met Ser Asp Leu Lys Ala Glu Lys195 200 205 Leu Val Met Phe Gln Arg Arg Tyr Tyr Lys Pro Gly Leu Leu LeuMet 210 215 220 Cys Phe Ile Leu Pro Thr Leu Val Pro Trp Tyr Cys Trp GlyGlu Thr 225 230 235 240 Phe Leu His Ser Leu Phe Val Ser Thr Phe Leu ArgTyr Thr Leu Val 245 250 255 Leu Asn Ala Thr Trp Leu Val Asn Ser Ala AlaHis Leu Tyr Gly Tyr 260 265 270 Arg Pro Tyr Asp Lys Asn Ile Gln Ser ArgGlu Asn Ile Leu Val Ser 275 280 285 Leu Gly Ser Val Gly Glu Gly Phe HisAsn Tyr His His Ala Phe Pro 290 295 300 Tyr Asp Tyr Ser Ala Ser Glu TyrArg Trp His Ile Asn Phe Thr Thr 305 310 315 320 Phe Phe Ile Asp Cys MetAla Ala Leu Gly Leu Ala Tyr Asp Arg Lys 325 330 335 Lys Val Ser Lys AlaAla Val Leu Ala Arg Ile Lys Arg Thr Gly Asp 340 345 350 Gly Ser His LysSer Ser 355 9 357 PRT Gallus gallus 9 Met Pro Ala His Leu Leu Gln GluGlu Glu Phe Ser Ser Ala Ser Ser 1 5 10 15 Thr Thr Thr Val Thr Ser ArgVal Thr Lys Asn Gly Asn Val Ile Met 20 25 30 Glu Lys Asp Leu Leu Asn HisAsp Asp Val Ala Ala Glu Arg Gly Met 35 40 45 Val Asp Asp Leu Phe Asp GluThr Tyr Arg Glu Lys Glu Gly Pro Lys 50 55 60 Pro Pro Leu Arg Tyr Val TrpArg Asn Ile Ile Leu Met Ser Leu Leu 65 70 75 80 His Leu Gly Ala Ile IleGly Leu Thr Leu Ile Pro Ser Ala Lys Ile 85 90 95 Gln Thr Leu Ala Trp AlaIle Leu Cys Phe Val Leu Ser Ala Leu Gly 100 105 110 Ile Thr Ala Gly SerHis Arg Leu Trp Ser His Arg Ser Tyr Lys Ala 115 120 125 Thr Leu Pro LeuArg Ile Phe Leu Thr Ile Ala Asn Ser Met Ala Phe 130 135 140 Gln Asn AspIle Tyr Glu Trp Ala Arg Asp His Arg Val His His Lys 145 150 155 160 PheSer Glu Thr His Ala Asp Pro His Asn Ala Met Arg Gly Tyr Phe 165 170 175Phe Ser His Met Ala Trp Leu Leu Val Arg Lys His Pro Asp Val Ile 180 185190 Glu Lys Gly Gln Lys Leu Asp Leu Ser Asp Leu Lys Ala Asp Lys Val 195200 205 Val Met Phe Gln Arg Arg Tyr Tyr Lys Pro Ser Val Val Leu Leu Cys210 215 220 Phe Thr Leu Pro Thr Leu Val Pro Trp Tyr Phe Trp Asp Glu SerIle 225 230 235 240 Ile Ile Ser Phe Phe Ile Pro Ala Ile Leu Arg Tyr ThrLeu Gly Leu 245 250 255 Asn Ala Thr Trp Leu Val Asn Ser Ala Ala His MetPhe Gly Asn Arg 260 265 270 Pro Tyr Asp Gln Asn Ile Asn Pro Arg Glu AsnPro Leu Val Ser Val 275 280 285 Gly Ala Leu Gly Glu Gly Phe His Asn TyrHis His Thr Phe Pro Tyr 290 295 300 Asp Tyr Ser Thr Ser Glu Phe Gly TrpArg Phe Asn Leu Thr Thr Ala 305 310 315 320 Phe Ile Asp Leu Met Cys LeuLeu Gly Leu Ala Ser Asp Arg Lys Lys 325 330 335 Val Ser Lys Glu Val IleLeu Ala Arg Lys Met Arg Thr Gly Asp Gly 340 345 350 Ser His Lys Ser Gly355

What is claimed is:
 1. An isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide comprising the sequence of SEQ ID NO:2.
 2. The nucleic acid of claim 1, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:2.
 3. An isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO:3 or a complete complement thereof.
 4. The nucleic acid of claim 3, wherein the nucleic acid consists of the nucleotide sequence of SEQ ID NO:3.
 5. The nucleic acid of claim 3, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO:1 or a complete complement thereof.
 6. An isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO:2 or the sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number 2167, wherein the percent identity is determined using the ALIGN program in the GCG software package, using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4, and wherein the polypeptide has fatty acid desaturase activity.
 7. The nucleic acid of claim 6, wherein the amino acid sequence is at least 90% identical to the sequence of SEQ ID NO:2 or the sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number
 2167. 8. The nucleic acid of claim 7, wherein the amino acid sequence is at least 95% identical to the sequence of SEQ ID NO:2 or the sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number
 2167. 9. The nucleic acid of claim 8, wherein the polypeptide comprises amino acid residues 51-295 of SEQ ID NO:2.
 10. The nucleic acid of claim 6, further comprising a sequence encoding a heterologous polypeptide.
 11. A vector comprising the nucleic acid of claim
 6. 12. The vector of claim 11, wherein the vector comprises nucleic acid sequences which regulate expression of a polypeptide encoded by the nucleic acid.
 13. A host cell comprising the vector of claim
 12. 14. The host cell of claim 13, which is a mammalian host cell.
 15. A method for producing a polypeptide, the method comprising culturing the host cell of claim 14 under conditions in which the nucleic acid is expressed to produce the polypeptide.
 16. An isolated nucleic acid comprising a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:3, wherein the percent identity is determined using the NBLAST program with a score of 100 and a word length of 12, and wherein the nucleotide sequence encodes a polypeptide having fatty acid desaturase activity.
 17. The nucleic acid of claim 16, wherein the nucleotide sequence is at least 90% identical to the nucleotide sequence of SEQ ID NO:3.
 18. The nucleic acid of claim 17, wherein the nucleotide sequence is at least 95% identical to the nucleotide sequence of SEQ ID NO:3.
 19. The nucleic acid of claim 16, wherein the polypeptide comprises amino acid residues 51-295 of SEQ ID NO:2.
 20. An isolated nucleic acid of at least 750 nucleotides in length comprising a nucleotide sequence that hybridizes to the sequence of SEQ ID NO:3 or the complete complement thereof under conditions of hybridization at 45° C. in 6.0×sodium chloride/sodium citrate (SSC) followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C., wherein the nucleotide sequence encodes a polypeptide having fatty acid desaturase activity.
 21. The nucleic acid of claim 20 wherein the nucleic acid comprises at least 800 nucleotides.
 22. The nucleic acid of claim 21, wherein the nucleic acid comprises at least 1000 nucleotides.
 23. The nucleic acid of claim 22, wherein the nucleic acid comprises at least 1200 nucleotides.
 24. The nucleic acid of claim 23, wherein the nucleic acid comprises at least 1400 nucleotides. 